Phycobiliprotein Lyases: Structure of Reconstitution

Phycobiliprotein Lyases:

Structure of Reconstitution Products and Mechanistic Studies

Jun-Ming Tu

2008 MUNICH

Phycobiliprotein Lyases:

Structure of Reconstitution Products and Mechanistic Studies

Jun-Ming Tu

Dissertation

zur Erlangung des Doktorgrades

der Fakultät für Biologie

der Ludwig-Maximilians-Universität

München

Submitted

by

Jun-Ming Tu

2008 MUNICH

1. Referee: Prof. Dr. Hugo Scheer 2. Referee: Prof. Dr. Lutz Eichacker Date of oral defense: October 29, 2008

Publications: Tu,J.M., Kupka,M., Böhm,S., Plöscher,M., Eichacker,L., Zhao,K.H., and Scheer, H. (2008) Intermediate binding of phycocyanobilin to the lyase, CpeS1, and transfer to apoprotein. Photosynth.Res. 95:163-168. Zhao, K. H., Su, P., Li, J., Tu, J.M., Zhou, M., Bubenzer, C. and Scheer, H. (2006) Chromophore attachment to phycobiliprotein beta-subunits: phycocyanobilin: cysteine-beta84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena sp. PCC7120. J. Biol. Chem. 281: 8573-8581. Zhao,K.H., Su,P., Tu,J.M., Wang,X., Liu,H., Plöscher,M., Eichacker,L., Yang,B., Zhou,M. and Scheer, H. (2007) Phycobilin:cysteine-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins. Proc Natl Acad Sci USA, 104:14300-14305. Zhao,K.H., Zhang,J., Tu,J.M., Böhm,S., Plöscher,M., Eichacker,L., Bubenzer,C., Scheer, H., Wang,X., and Zhou,M. (2007) Lyase activities of CpcS and CpcT-like proteins from Nostoc PCC7120, and sequential reconstitution of binding sites of phycoerythrocyanin and phycocyanin β-subunits. J. Biol. Chem. 282:34093-34103

Acknowledgements This work was performed at the laboratory of Prof. Dr. H. Scheer in the Department I of the Botanical Institute of the Ludwig-Maximilians-University in Munich, Germany and at the laboratory of Prof. Dr.K-H. Zhao in College of Environmental Science and Engineering, Huazhong University of Science and Technology in Wuhan, PR China I am very much grateful to my supervisor Prof. Dr. H. Scheer for theoretical and practical supervision during the work. I would like to thank him for inviting me into his group, for giving me an opportunity to conduct my PhD research in his laboratory and for excellent working conditions. I would like to acknowledge him for his help, continuous support and valuable discussions throughout my PhD study. I am thankful for the advises that he gave me, for careful reading and correction of this thesis and for his helpful comments. I also appreciate a lot his optimism and never-ending enthusiasm. I am thankful to my supervisor in PR China Prof. Dr. K-H.Zhao. Thanks for his support, care, for teaching me at the beginning of my studies in Huazhong University of Science and Technology, for introducing me into molecular biology and providing all of plasmids for this work. My sincere thanks go to Dr. Stephan Böhm for his excellent technical help, support, patience, care and for interesting talks about life. I am grateful to him for his bright personality and for helping me to better understand people of his country My special thanks to Dr. Zhen-Hua Cui for her help, for introducing me into the lab life, for scientific and personal support since my very first days in the lab, for nice discussions and for good time in and outside the lab. Many thanks to Prof. Dr. Lutz Eichacker and Dr. Matthias Plöscher (LMU, Biology Department I, Munich) for the ESI-MS measurements, and to Dr. Rainer Haessner (Technical University Munich, Germany) for NMR measurements. I would like to thank all graduate students and post-docs who used to work or are currently working in this group for their friendly and helpful collaboration, for creating a pleasant working atmosphere and for a nice time. My dear parents, sister and my wife H-X Tong; many, many thanks for your constant and genuine love, care, encouragement, understanding, inspiration and support at each step of my life. Thank you very much! Last but not least, I gratefully acknowledge the Chinese Scholarship Council for a doctoral scholarship.

Abbreviations A absorption a.u. arbitrary unit APC allophycocyanin ApcA apoprotein of allophycocyanin α-subunit ApcA2 apoprotein of allophycocyanin α-subunit ApcD apoprotein of allophycocyanin α-subunit ApcB apoprotein of allophycocyanin β-subunit ApcF apoprotein of allophycocyanin β-subunit APS ammonium persulfate bp base pair BSA bovine serum albumin BV IXa biliverdin IXa CD circular dichroism Cpc A apoprotein of C-Phycocyanin α-subunit CpcB apoprotein of C-Phycocyanin β-subunit CpcE, CpcF subunits of Phycocyanobilin-α-Phycocyanin-lyase DNA deoxyribonucleic acid DMSO dimethyl sulfoxide E. coli Escherichia coli EDTA ethylenediaminetetraacetic acid ESI-MS electronspray ionization mass spectrometry NR ferredoxin NADP+ oxidoreductase hr hour(s) IPTG isopropyl β-D-thiogalactoside k rate constant [s-1] kDa kiloDalton LB Luria-Bertani medium ME 2-mercaptoethanol MHz Megahertz min minute MS mass spectrometry MW molecular weight m/z mass per charge nm nanometer NMR nuclear magnetic resonance NOESY nuclear Overhauser and Exchange Spectroscopy ORF open reading frame PAGE polyacrylamide gel electrophoresis PBS phycobilisome PC phycocyanin PCB phycocyanobilin PCR polymerase chain reaction

PE phycoerythrin PEB phycoerythrobilin PEC phycoerythrocyanin PecA apoprotein of phycoerythrocyanin α-subunit PecB apoprotein of phycoerythrocyanin β-subunit PecE and PecF phycoviolobilin-α84-cystein-lyase-isomerase subunits PFB phytochromobilin PK protein kinases PSI photosystem I PSII photosystem II PVB phycoviolobilin (4,5-Dihydro-mesobiliverdin) rpm revolutions per minute SDS sodium dodecyl sulfate TBE tris-borate-EDTA buffer TE Tris-EDTA TEMED N, N, N’, N’-tetramethylethylene diamine TFA trifluoroacetic acid TLC thin layer chromatography tr retention time Tris tris-(hydroxymethyl)-aminomethane TritonX-100 alkylphenyl polyethylenglycol WT wild type v/v volume per volume QVis/UV ratio of maximum absorption in the visible (Vis) and the

ultraviolet (UV) spectral region w/v weight per volume

Table of Contents

TABLE OF CONTENTS 1: Introduction...................................................................................................................................1

1.1 Structures of phycobilisomes ..............................................................................................1 1.2 Biosynthesis of phycobilins ................................................................................................6 1.3 Chromophore attachment ....................................................................................................7

1.3.1 Spontaneous attachment...........................................................................................8 1.3.2. Autocatalytic attachment .........................................................................................9 1.3.3. E/F-type lyases ........................................................................................................9 1.3.4. Cyanobacterial S/U-type lyases ............................................................................10 1.3.5. Cyanobacterial T-Type lyases................................................................................11

1.4 Lyase mechanisms.............................................................................................................11 1.5 Applications ......................................................................................................................13 1.6 Thesis plan ........................................................................................................................14

2: Materials and Methods................................................................................................................16 2.1 Materials ...........................................................................................................................16

2.1.1 Organisms ..............................................................................................................16 2.1.2 Vectors....................................................................................................................16 2.1.3 Plasmids .................................................................................................................16 2.1.3 Chemicals...............................................................................................................17 2.1.4 Technical devices ...................................................................................................20

2.2 Methods.............................................................................................................................22 2.2.1 General molecular biological methods...................................................................22 2.2.2 Protein isolation .....................................................................................................22 2.2.3 Pigment isolation....................................................................................................25 2.2.4 Phycobiliprotein reconstitution/preparation ...........................................................27 2.2.5 Lyase CpcS (Alr0617) reaction assay ....................................................................29 2.2.6 Chromophore adduct assay ....................................................................................29 2.2.7 Spectroscopy ..........................................................................................................30 2.2.8 Websites .................................................................................................................31

3: Results and Discussion ...............................................................................................................32 3.1 Structure analysis of reconstituted holo-PecA ..................................................................32

3.1.1 Reconstitution principle of holo-PecA...................................................................32 3.1.2 Expression and Purification of the holo-PecA .......................................................33 3.1.3 Circular Dichroism Spectroscopy ..........................................................................35 3.1.4 Photoisomerization of α-PEC ................................................................................36 3.1.5 HPLC–ESI-MS analysis of chromopeptides from holo-PecA ...............................37 3.1.6 NMR analysis of peptic chromopeptides from holo-PecA.....................................39 3.1.7 Discussion ..............................................................................................................42

3.2 Structure analysis of reconstituted holo-CpcA..................................................................45 3.2.1 Reconstitation principle of holo-CpcA ..................................................................45 3.2.2 Expression and Purification of the holo-CpcA.......................................................46 3.2.3 Circular Dichroism Spectroscopy ..........................................................................48 3.2.4 HPLC–ESI-MS analysis of chromopeptides from holo-CpcA ..............................48

Table of Contents

3.2.5 NMR analysis of peptic chromopeptides from holo-CpcA....................................50 3.2.6 Discussion ..............................................................................................................53

3.3 Structure analysis of reconstituted subunits of allophycocyanin.......................................55 3.3.1 Reconstitution principle of subunits of allophycocyanin .......................................55 3.3.2 Expression and Purification of the subunits of allophycocyanin ...........................56 3.3.3 Circular Dichroism Spectroscopy ..........................................................................58 3.3.4 Chromophore analyses of reconstitution products .................................................59 3.3.5 HPLC–ESI-MS analysis of chromopeptides from reconstituted subunits of allophycocyanin ......................................................................61 3.3.6 NMR analysis of peptic chromopeptides from reconstituted subunits of allophycocyanin ......................................................................63 3.3.7 Discussion ..............................................................................................................67

3.4 Structure analysis of reconstitution β-subunit of C-phycocyanin .....................................71 3.4.1 Reconstitution principle of β-subunit of C-phycocyanin .......................................71 3.4.2 Expression and Purification of the β-subunits of C-phycocyanin..........................72 3.4.3 Circular Dichroism Spectroscopy ..........................................................................73 3.4.4 Chromophore analyses of reconstitution products .................................................74 3.4.5 HPLC–ESI-MS analysis of chromopeptides from reconstituted β-subunits of C-phycocyanin.....................................................................76 3.4.6 NMR analysis of peptic chromopeptides from reconstituted β subunits of C-phycocyanin .....................................................................78 3.4.7 Discussion ...........................................................................................................82

3.5 Structure analysis of reconstituted β-subunits of phycoerythrocyanin..............................85 3.5.1 Reconstitution principle of β-subunit of phycoerythrocyanin................................85 3.5.2 Expression and Purification of the β-subunits of phycoerythrocyanin ..................85 3.5.3 Circular Dichroism Spectroscopy ..........................................................................87 3.5.4 Chromophore analyses of reconstitution products .................................................88 3.5.5 HPLC–ESI-MS analysis of chromopeptides from reconstituted β-subunits of phycoerythrocyanin .............................................................90 3.5.6 NMR analysis of peptic chromopeptides from reconstituted β-subunits of phycoerythrocyanin .............................................................92 3.5.7 Discussion ..............................................................................................................96

3.6 Structure analysis of imidazole-bilin adducts ...................................................................98 3.6.1 Binding of PCB to CpcS ........................................................................................98 3.6.2 Imidazole binding PCB..........................................................................................99 3.6.3 Chromophore transfer from CpcS-PCB to CpcB(C155I) ....................................102 3.6.4 Chromophore transfer from imidazole-bilin adducts to CpcB(C155I), CpcS or mercaptoethanol ..................................................................103 3.6.5 HPLC–ESI-MS analysis of imidazole-bilin adducts............................................106 3.6.6 NMR analysis of imidazole-bilin adducts ............................................................107 3.6.7 Discussion ............................................................................................................110

3.7 Structure analysis of mercaptoethanol-bilin adducts.......................................................113 3.7.1 Mercaptoethanol binding PCB catalyzed by CpcS ..............................................113 3.7.2 Spontaneous binding of mercaptoethanol to PCB................................................115

Table of Contents

3.7.3 Structure analysis of mercaptoethanol-bilin adducts............................................117 3.7.4 Chromophore transfer from CpcS-PCB to ME ....................................................122 3.7.5 Chromophore transfer from bilin adducts to CpcB(C155I) .................................122 3.7.6 Discussion ............................................................................................................124

3.8 Attachmentof a locked chromophore, 15Za-PCB ...........................................................127 3.8.1 Structure of 15Za-PCB.........................................................................................127 3.8.2 Absorption and extinction coefficient of 15Za-PCB............................................127 3.8.3 Chromophore binding assay.................................................................................128 3.8.4 Denaturation of the 15Za-PCB addition products ................................................130 3.8.5 Chromophore reaction with Zn2+ .........................................................................131 3.8.6 The effection of pH on chromophore spectra.......................................................132 3.8.7 Conclusion ...........................................................................................................133

3.9 Test of a protein with Heat-like repeats, DOHH, for lyase activity ................................134 4: Concluding Discussion .............................................................................................................136

4.1 A multi-plasmidic expression system..............................................................................136 4.2 A multi-step analytic system ...........................................................................................137 4.3 E/F-type lyase catalyzed attachment ...............................................................................139 4.4 S (/U)-type lyase catalyzed attachment...........................................................................139 4.5 T-type lyase catalytic attachment ....................................................................................141 4.6 Imidazole-bilin adducts...................................................................................................143 4.7 Mercaptoethanol-bilin adducts........................................................................................144 4.8 Some interesting observations as by-products of the experiments..................................145 4.9 Future prospects ..............................................................................................................146

5: Summary .................................................................................................................................148 6: References..............................................................................................................................151 Curriculum Vitae ..........................................................................................................................172

1. Introduction 1

1: Introduction

1.1 Structures of phycobilisomes

Phycobilisomes (PBSs) are multimeric highly organized protein complexes that

harvest light in the wavelength range from 480 to 650 nm in the “chlorophyll gap” and

may constitute 50% of the soluble proteins in cyanobacterial and red algal cells.

Contrary to light harvesting antennae, LHCII, of higher plants which are located within

the thylakoid membrane, the PBS of cyanobacteria are peripherally associated with

the outer cytoplasmic surface of the photosynthetic membranes (Glazer et al., 1983,

1985) (Fig. 1-1). PBSs are not only antenna complexes for light harvesting, but can

also be used as storage materials for carbon and nitrogen (Bryant, 1987).

Fig. 1-1: Major respiratory and photosynthetic electron transport components of cyanobacteria (Bryant, 1994). A,B,C = core of allophycocyanin; D-G: rod of phycocyanin (D,E) and phycoerythrin or phycoerythrocyanin (F,G).

These PBSs are composed of two types of proteins: colored phycobiliproteins (PBPs),

which absorb and transmit light energy to the photosynthetic reaction centers, and

non-pigmented linker proteins, which organize the former into the PBSs and modulate

their absorptions (Glazer, 1989).

The non-pigmented linker proteins are integrated into the PBS structure (Fig.1-2).

1. Introduction 2

These polypeptides with molecular masses from 8 kDa to 120 kDa serve several

functions in the PBSs. They help stabilizing the PBS structure, determine the

positions of specific PBPs in the complex, facilitate assembly of PBP-containing

substructures, modulate the absorption characteristics of the PBPs to promote

unidirectional transfer of energy within the PBSs and from PBSs to chlorophylls of the

photosynthetic reaction centres, and physically link the entire complex to the

photosynthetic membranes (Glazer et al., 1983, 1985, 1988, 1989; Bryant, 1991;

Grossman et al., 1993). Linker proteins can be divided into four groups: rod core

linkers (LRC) that attach the peripheral rods to the PBS core, rod linkers like LR10, LR

33,

LR35(the superscript indicates the molecular weight) that associate phycocyanin (PC)

or phycoerythrin (PE) substructures into rod segments, the small core linkers like LC8,

that are associated with trimeric allophycocyanin (APC) at the peripheries of the core

cylinders, and the core-membrane linker like LCM99 that acts in the organisation of the

PBS core, in the PBS attachment to the membrane and due to its chromophore, also

as the major terminal energy emitter to PSII (Glazer, 1989; Capuano et al., 1991;

Sidler, 1994). The latter one was suggested to couple PBSs to the membrane via a

domain called the PB-loop, but deletion mutagenesis of this domain that has been

proposed to be a transmembrane domain, did not show any changes in the

association of PBSs with thylakoid membranes in the mutant strain (Ajlani and

Vernotte, 1998; Piven, 2006 ). Most of the linker proteins are colorless, but at least two

of them also carry covalently bound chromophores, namely, the aforementioned

core-membrane linker Lcm (= PCB-ApcE), and the γ-subunits of class II and some

class I phycoerythrins (PEs) (Scheer and Zhao, 2008).

Fig. 1-2: Schematic structure of PBSs of Synechocystis sp. PCC 6803. PC – phycocyanin, APC – allophycocyanin, LCM– core -membrane linker, LC

–core linker, LRC

– rod

corelinker, LR – rod linker (Piven, 2006)

1. Introduction 3

PBPs are categorized into three types according to their absorption maximum in the

visible range (Table 1-1), those of high energy (PE or phycoerythrocyanin (PEC)),

intermediate energy (PC), and low energy (APC) (Tab.1-1). Energy will flow from

highest- to lowest-energy pigments and this is how the PBSs are organized (Fig. 1-1).

The details of PBS structure are quite variable. However, a common theme is that the

PBS has a central ‘‘core’’ composed of APCs and specific linker polypeptides that is

situated in proximity to the thylakoid membrane and photosystem II, where chlorophyll

a is located. Radiating out from the core are a number of ‘‘rod’’ elements composed of

PCs (and often also PEs and other PBPs) together with their associated linker

polypeptides. The entire assemblage typically has a molecular mass in the range from

7 to 15 MDa. Cyanobacterial PBSs are typically hemidiscoidal in overall form, with

diameters in the range of 32–70 nm (Grossman 1993). In hemidiscoidal PBSs, there

are generally six rods, and three cylinders in their core.

Cyanobacterial and red-algal biliproteins are generally trimers of an α/β -heterodimer.

α and β subunits are closely related proteins, carrying 1–4 covalently bound

chromophores, the phycobilins (Fig. 1-3).

Fig. 1-3: Levels of PBS association as seen in the crystallographic unit cell of Tv-PC620. The α and β subunits are shown in Ca traces for simplicity (yellow and blue, respectively). Application of crystallographic symmetry reconstructs a number of physiologically important aggregation states: the (α β)3 trimer (the top of each ring), the (α β)6 hexamer disk (the bottom trimer is directly below the top trimer and is thus superimposed). Notice that the three PCB cofactors are separated spatially: α84 PCBs (red) are buried in the (α β) interface of the trimer, β84 PCBs (green) are all located within the trimer and hexamer disk and probably associate with linker proteins and β155 PCBs (pink) are all located on the circumferential surface of the disks and may help in energy transfer between disks and between rods (Adir, 2005).

1. Introduction 4

Table 1-1: The visible absorption maxima of various phycobiloiproteins (after Coyler et al., 2005)

Phycobiliprotein Maxima of visible absorption [nm]

Color

Phycoerythrin (PE) 540-570 orange-red Phycoerythrocyanin (PEC) 570-590 purple Phycocyanin (PC) 610-620 blue Allophycocyanin (APC) 650-655 blue-green

Table 1-2: Phycobilin binding sites and types of various phycobiliproteins

Binding sites of bilins Biliproteins

α-75 α-84* α-140 β-50/60 β-84* β-155

Allophycocyanin PCB PCB

C-Phycocyanin PCB PCB PCB

Phycoerythrocyanin PVB PCB PCB

R-Phycocyanin PEB PCB PEB

R-Phycocyanina PUB PCB PEB

Phycocyanin

(WH8501)

PUB PCB PCB

C-Phycoerythrin PEB PEB PEB PEB PEB

CU-Phycoerythrinb PEB PUB PUB PEB PEB

CU-Phycoerythrinc PUB PEB PEB PUB PEB PEB

After Frank et al. 1978; Sidler et al. 1981; Füglistaller et al. 1983; Klotz et al. 1985; Sidler et al.1986; Ong et al. 1987; Swanson et al. 1991

* Consensus numbering; a) Synechoccus sp WH8102 (unpublished); b) Synechococcus sp. WH8103 PE(I); c) Synechococcus sp. WH8020 PE(II) (Boehm, 2006)

Individual PBP subunits carry 1-4 chromophores. The binding sites and types of bilins

associated with the subunits of various PBPs are summarized in Table 1-2.

1. Introduction 5

31-Cys-PUB

31-Cys-PEB

31-Cys-PVB

Free PUB(hypothetical)

Free PEB

Free PVB(hypothetical)

N N

COOH

COOH

HN

O

H

H

NH

O

RHH

A

BC

D

N N

COOH

COOH

H

NH

O

N

O

RHH H

A

BC

D

NH

N N

COOH

COOH

O

H

N

O

RHH

A

BC

D

NH

N N

COOH

COOH

HN

O O

H

H

RHA

BC

D

NN NH

NH

COO-

OO

H

SCys

COO-

A

BC

D

NN NH

NH

COO-

OO

H

SCys

COO-

A

BC

D

NN NH

NH

COO-

OO

H

SCys

COO-

A

BC

D

NNNH

NH

COO-

OO

H

SCys

COO-

A

BC

D

Free PCB 31-Cys-PCB

33

1

Fig. 1-4: Structures of free and protein-bound bilins. Structures of PCB, PVB (not known as free chromophore), PEB, PUB(not known as free chromophore) in their free forms (left) and bound at C-31 to the apoprotein via cysteine thioether bond. Free chromophores prefer a cyclic-helical conformation, bound ones have extended comformations in the native biliproteins (Boehm et al, 2007).

With the number of binding sites increasing from APC to PC and PEC and further to

PEs (and also cryptophyte biliproteins that are not organized in phycobilisomes

(Sidler, 1994)), the α- and β-subunits of phycobiliproteins carry at least one linear

tetrapyrrole chromophore (bilin) called phycocyanobilin (PCB), phycoviolobilin (PVB),

1. Introduction 6

phycoerythrobilin (PEB) or phycourobilin (PUB) which is covalently attached to

cysteines of the apoprotein via a thioether bond to C-31 on ring A (Fig. 1-4) and in

some cases by an additional thioether bond to C-181 on ring D (Sidler, 1994).

1.2 Biosynthesis of phycobilins

Bilin is the collective generic name for linear tetrapyrroles. The major bilins in nature

are biliverdin (BV), bilirubin (BR), PCB, PEB and phytochromobilin (PΦB), they are all

derived from heme. The biosynthesis is summarized in Fig1-5.

Fig. 1-5: Chemical structures and biosynthetic pathways of chlorophylls, heme and bilins. BV is synthesized from 5-aminolevulinic acid (ALA) via heme. BV is further reduced to produce PΦB in plants, PEB and PCB in cyanobacterial and algae and BR in animals by the action of different bilin reductase. Note that no obvious sequence similarity was detected between BV reductases (BVR and bvdR) and other ferredoxin-dependent bilin reductases (HY2, PcyA, PebA and PebB) (Kohchi et al., 2005).

Heme, which contains iron, is synthesized from protoheme IX by ferrochelatase. Heme

is an essential prosthetic group in many reduction/oxidation processes. Heme is

cleaved to BV IXα by heme oxygenase; this is a degradation process in animals but for

photosynthetic organisms this step is a biosynthetic process (Muramoto et al. 1999).

Further reduced bilins are synthesized from BV IXα by the action of different

ferredoxin- dependent bilin reductases (FDBRs) (Kohchi et al. 2001; Frankenberg et al.

1. Introduction 7

2003; Dammey et al.,2008), a new family of radical enzymes.

The reduction to phycocyanobilin is catalyzed by the FDBR phycocyanobilin:ferredoxin

oxidoreductase,PcyA, that carries out two reductions, at ring A and the 18-vinyl group

(Frankenberg et al., 2003). PΦB reductase (Hy2) catalyzes only reduction of ring A,

leading to PΦB, the typical chromophore of plant phytochromes (Kohchi et al., 2001;

Gambetta et al., 2001), while a dedicated reductase catalyzing only reduction of the

18-vinyl group would yield mesobiliverdin (MBV), one of the chromophores of

cryptophyte biliproteins.

The biosynthesis of PEB requires two subsequent two-electron reductions, each step

being catalyzed by one FDBR. The first reaction in PEB biosynthesis is the reduction of

the ∆15,16-double bond of BV IXα by 15,16-dihydrobiliverdin:ferredoxin

oxidoreductase (PebA). This reaction shortens the conjugated π-electron system,

thereby blue-shifting the absorption maxima of the linear tetrapyrrole. The second

FDBR, PEB:ferredoxin oxidoreductase (PebB), then reduces the A-ring

2,3,31,32-diene structure of 15,16-dihydrobiliverdin to yield PEB (Dammeyer et al.,

2006). A single subunit reductase that carries out both steps has been characterized

(Dammeyer et al., 2008).

1.3 Chromophore attachment

The final step in phycobiliprotein biosynthesis is the covalent chromophore attachment

to the apoprotein. Heterodimeric lyases (CpcE/CpcF and PecE/pecF) that catalyzes

the covalent attachment of PCB to cysteine-αC84 of CPC, and of PVB to cysteine-α84

of PEC, respectively, were charaterized before the beginning of this thesis. During,

and in part in this thesis, the S/U-type and T-type lyase were charaterized by Zhao et

al. (2006a, 2007a,b) and Shen et al. (2006, 2008a). Currently, there are five

characterized modes of chromophore attachment (Tab. 1-3).

1. Introduction 8

Table 1-3: Modes of chromophore attachment in biliproteins

Mode Biliprotein

1. Spontaneous attachment Most biliproteins in the absence of lyase (Arciero et al., 1988; Fairchild and Glazer, 1994a; Schluchter and Glazer, 1999; Zhao et al.,2007a, b; Scheer and Zhao, 2008; Boehm et al., 2007)

2. Autocatalytic attachment Phytochromes (Wu and Lagarias, 2000; Zhao et al., 2004; Bhoo et al., 1997; Lamparter, 2004; Wagner et al., 2005)

LCM (=PCB-ApcE) (Zhao et al., 2005)

3.Catalyzed by E/F-type lyases

PCB-CpcA (Zhou et al., 1992; Tooley et al., 2001)

PVB-PecA (Zhao et al., 2000; Tooley et al., 2002)

4.Catalyzed by cyanobacterial S/U-type lyases

CpcB-C84-PCB (Shen et al., 2004; Zhao et al., 2006a; Shen et al., 2008; Saunée et al., 2008)

PecB-C84-PCB (Zhao et al., 2006a)

CpeA-C84-PEB, CpeB-C84-PEB (Zhao et al., 2007a)

ApcA,B,D,F-PCB (Zhao et al.,2007a; Saunée et al., 2008)

5.Catalyzed by cyanobacterial T-Type lyases

CpcB-C155-PCB ( Shen et al., 2006; Zhao et al., 2007b)

PecB-C155-PCB (Zhao et al., 2007b)

1.3.1 Spontaneous attachment

Most apoproteins can bind phycobilins (PCB, PEB) spontaneously in vitro. This

process is very frequently observed, but is of low fidelity and generally leads to product

mixtures containing oxidation products and incorrect stereochemistry of the adduct

(Arciero et al., 1988; Fairchild and Glazer, 1994a; Schluchter and Glazer, 1999;

Boehm et al., 2007). Spontaneous attachment is a considerable problem in binding

studies, especially in vitro, because it interferes with lyase assays and is not easily

distinguished from truly autocatalytic lyase activities (Schluchter and Bryant, 2002;

Zhao et al., 2007; Böhm et al., 2007).

1. Introduction 9

Scheer and Zhao (2008) analyzed important clues for understanding and

distinguishing correct chromophore binding and spontaneous binding. Firstly, many, if

not all, apoproteins can form thioether bonds with suitable chromophores. Secondly,

the bonds are formed with the correct cysteines, namely, only those located at the

native binding sites, indicating site-specific interactions of the chromophores with the

binding pockets. This is further emphasized by the non-covalent, yet (largely)

functional binding of chromophores in mutants that lack the chromophore binding

cysteine residue (Lamparter and Michael, 2005; Gindt et al., 1992; Shen et al., 2008),

or the non-covalent binding of modified chromophores (Inomata et al., 2006).

1.3.2. Autocatalytic attachment

Autocatalytic attachment has been reported for ApcA from two cyanobacteria,

including Anabaena PCC7120 (Hu et al., 2006; Zhao et al., 2007). In our view, the

product differed distinctly in its absorption, fluorescence and circular dichroism from

isolated native α-APC, this is therefore an incorrect attachment, spontaneous rather

than a true autocatalytic attachment. Currently, autocatalytic binding in

phycobiliproteins has only been found for ApcE that could be reconstituted with PCB to

give native-like LCM. (Zhao et al., 2005), and for the various phytochromes (Wu and

Lagarias, 2000). Both the chromophore carrying and the lyase domains reside in the

N-terminal region of this large protein. Phytochromes have two alternative binding

sites; both are chromophorylated autocatalytically. The autocatalytic chromophore

attachment in phytochromes requires the co-action of domains that are near the two

alternative binding sites (Zhao et al., 2004; Bhoo et al., 1997; Lamparter et al., 2004).

1.3.3. E/F-type lyases

Up to date, two E/F-type lyases (CpcE/CpcF and PecE/PecF) have been studied. A

heterodimeric lyase (CpcE/F) from Synechococcus PCC 7002 (Zhou et al., 1992) is

the first phycobiliprotein lyase that was identified by an in vitro assay. Tooley et al.

(2001) also proved this conclusion by heterologous in vivo reconstitution in E. coli.

CpcE/F is required for correct binding of PCB to Cys-α84, and catalyzes both the

forward (binding) and the reverse (releasing) reaction (Fairchild et al., 1992). It also

catalyzes the addition of PEB to apo-α-CPC (CpcA), but with reduced affinity and

1. Introduction 10

kinetics compared to PCB (Fairchild and Glazer, 1994b).

The heterodimeric lyase/isomerase (PecE/PecF) catalyzes both the covalent

attachment of PCB at Cys-α84 of PecA and the concurrent isomerization of the

molecule to PVB. This conclusion was reached by in vitro and in vivo reconstitution by

Tooley et al. (2002, in vivo), Zhao et al. (2000, in vitro) and Storf et al. (2001, in vitro).

The data indicate high protein specificity combined with a high site-specificity for the

E/F-type lyases.

Zhao et al. (2002) studied the cofactor requirements and enzyme kinetics of the

novel, dual-action enzyme, the isomerizing phycoviolobilin

phycoviolobilin:phycoerythrocyanin-Cysα84 lyase (PVB:PEC-lyase) from M.

laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA,

the apo-α-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. A

mechanism has been proposal by Boehm et al.(2007). A similar reaction sequence

would generate bound PUB from free PEB, lyases for this process have been

proposed by Six et al. (2007).

1.3.4. Cyanobacterial S/U-type lyases

Shen et al.(2004) presented first evidence for new types of lyases in Synechococcus

PCC7002 at a meeting. One of them, cpcS, has very high sequence similarity to cpeS

in Fremyella diplosiphon, this gene is located on an operon (cpeCDESTR) that was

sequenced, and related to the biogenesis of phycoerythrin (Cobley et al., 2002).

The possible function of CpcS has been rapidly characterized thereafter. In vitro and in

E. coli, it can catalyses chromophore attachment to all binding sites of APCs, to β84 of

CPCs and PECs, and even to some binding sites (α84 and β84) of CPEs (Zhao et al.,

2007a; Zhao et al., 2006a; Saunée et al., 2008; Shen et al., 2008). These data

indicated that S-type lyases are near-universal lyases for cysteine-84 binding sites in

cyanobacterial phycobiliproteins. Notable expections are chromophore attachments to

α84 of CPC, PEC that were catalyzed by E/F type lyases(see section 1.3.3).

CpcS from Anabaena PCC 7120 (Zhao et al., 2006a; Zhao et al., 2007a) and CpcS

from Synechococcus PCC 7002 (Shen et al., 2008; Saunée et al., 2008), the two

1. Introduction 11

CpcS-type lyases studied so far seem to belong to two subtypes: CpcS from

Anabaena PCC 7120 acts as a monomeric, single subunit lyase. By contrast, CpcS

from another organism, Synechococcus PCC 7002, is inactive on its own, and requires

CpcU as a second subunit for activity. This functional heterogeneity is reflected by the

phylogenetic classification of the two subtypes into different groups (Ⅲ and Ⅰ,

respectively) of the complex S/U protein family (Shen et al., 2008).

1.3.5. Cyanobacterial T-type lyases

Synechococcus PCC7002 has yet another gene, cpcT, that is homologous to cpeT on

the Fremyella operon (Cobley et al., 2002); it codes for a third type (T-type) of lyase.

CpcT catalyzes the regiospecific PCB addition at Cys-155 of CpcB from

Synechococcus PCC7002 (Shen et al., 2006), and to Cys-155 of both β-CPC and β-

PEC in Anabaena PCC 7120 and M. laminosus (Zhao et al., 2007b). Since CpcB and

PecB are relatively closely related, these data indicate moderate protein specificity

combined with a high site-specificity for the T-type lyases.

Taking these findings together, in phycobilisome-containing cyanobacteria devoid of

PE, three lyases have been identified that are capable of attaching all phycobiliprotein

chromophores: CpcS(/U) attach PCB to Cys-β84 of PC and PEC, CpcT attach PCB to

Cys-β155 of PC and PEC , CpcE/F and PecE/F catalyze attachment to Cys-α84 of PC

and of PEC, respectively, and CpcS also attaches PCB to Cys-α84 and Cys-β84 of

APCs. Together with the autocatalytic chromophore attachment to ApcE, this would

allow for complete chromophorylation of the respective phycobilisomes. Note,

however, that there is presently only very little known on the sequence and the

regulation of the attachments.

1.4 Lyase mechanisms

Currently, there are very few mechanistic studies on biliprotein lyases published, and

they have focused on CpcE/F from Synechococcus PCC 7002 (Fairchild and Glazer,

1994b; Schluchter and Glazer, 1999), on CpcE/F and PecE/F from Mastigocladus

laminosus and Anabaena PCC7120 (Böhm et al., 2007), and on CpcS1 from

Anabaena PCC7120 (this thesis, Tu et al., 2008) that has been classified as an S-type

1. Introduction 12

lyase of group III (Shen et al., 2008). Most of this work has been published during the

time that this thesis has been prepared.

CpcS1 from Anabaena PCC7120 is a relatively simple system. It is active as a

monomer, and does not require cofactors (Zhao et al., 2007a; Zhao et al., 2006a). Tu

et al. (2008) have show that CpeS1 can bind PCB, as assayed by Ni2+ chelating affinity

chromatography. Binding is rapid, and the chromophore is bound in an extended

conformation similar to that in phycobiliproteins, but it is only poorly fluorescent. Upon

addition of apo-biliproteins, the chromophore is transferred to the latter much more

slowly (~1 h), indicating that chromophorylated CpeS1 is an intermediate in the

enzymatic reaction.

Fig. 1-6: Reaction scheme of the isomerizing E/F-type lyases (Scheer and Zhao, 2008) Non-covalent chromophore binding is indicated by broken lines, covalent binding by solid lines and coloration of both the chromophore and the protein. Intermediates (Pxxx) are named according to their absorption maxima at xxx nm.

The reaction catalyzed by the E/F-type lyases is more complex (Fig 1-6). The first

reaction step is addition of a fraction of the PCB (PCB*) immediately available for

binding to CpcE/F and subsequent transfer to apo-α-PC (CpcA) from Anabaena

PCC7120. The second, slower rate would depend on the rate of isomerization of the

bulk of the PCB to PCB*(Fairchild et al., 1992). PCB addition to the apoprotein by the

E-subunit is slow, but is accelerated if a complex of the protein substrate with the

1. Introduction 13

E-subunit is formed prior to chromophore addition, indicating that protein-protein

interactions are rate limiting. These studies (Böhm et al., 2007) with the E-subunit also

provided the first experimental evidence for a chaperone-like function that had

previously been discussed for lyases (Schluchter and Glazer, 1999). PecE can

transform a low absorption, low fluorescence spontaneous addition product, P645, to a

product P641 that has the high absorption and high fluorescence typical for native

biliproteins: P641 is the same product that is formed from PecA and PCB in the

presence of the E-subunit. Both lyase subunits are required for chromophore

attachment. The reaction has been studied in some detail with the isomerizing lyase,

PecE/F from Anabaena PCC 7120. The F-subunit alone is inactive, but carries the

isomerase activity for which a motif has been identified (Zhao et al., 2005); the

E-subunit alone catalyzes only the PCB attachment without isomerization (Scheer and

Zhao, 2008).

1.5 Applications

The first “application” of lyase-based reconstitution systems is studies on biliprotein

function. Tooley et al. 2001 first attempted to use complete biosynthesis of holo-CpcA

from heme in E. coli. The lyases, for the first time, make it possible to modify the

chromophore and protein separately, then linking them covalently by the use of the

appropriate lyase(s). This reconstitution system consists of 1) the apoprotein, 2) the

phycobilin biosynthesis enzyme, and 3) the lyase(s) (Tooley and Glazer, 2002; Tooley

et al., 2001; Saunée et al., 2008; Zhao et al., 2006a, 2007a, 2007b).

The second emerging application in basic research is studies on protein folding, where

covalently bound chromophores in a native system are desirable to avoid artifacts. In

particular, fluorescence has been proposed as an indicator for the protein dynamics,

and absorption and Vis-CD as indicators for the tertiary structure (Kupka and Scheer,

2008; Ma et al., 2007).

The third application is the use of PVB-containing α-PEC-(fusion)-proteins as a light

switch. The native protein environment enables the PVB chromophore to be switched

between two states with different absorption and vastly different fluorescence

characteristics: the 15Z-isomer is highly fluorescent, the 15E-isomer is practically

1. Introduction 14

non-fluorescent (Zhao et al., 1995). Alone or in combination with acceptor proteins,

this might allow for use in new high-resolution light microscopy techniques (Walter et

al., 2008).

The fourth application is as fluorescene label. Phycobiliprotein properties (high molar

extinction coefficients, high fluorescence quantum yields, large Stokes shifts, high

oligomer stability and relatively high photostability) make them potentially powerful and

highly sensitive fluorescent reagents. They can serve as labels for antibodies,

receptors and other biological molecules in a fluorescence-activated cell sorter and be

used in immunolablling experiments and fluorescence microscopy or diagnostics

(Spolaore, et al., 2006). In addition, because of the high molar absorptivity of these

proteins at visible wavelengths, they are convenient markers in such applications as

gel electrophoresis, isoelectric focusing and gel exclusion chromatography.

A final commerical application is as natural pigments. The primary potential of these

molecules seems to be as natural dyes, but an increasing number of investigations

have indicated health-promoting properties and a range of pharmaceutical

applications. The most important current application of phycocyanin is as colorant in

food (chewing gums, dairy products, ice sherbets, jellies etc) and cosmetics such as

lipsticks and eyeliners in Japan, Thailand and China, replacing current synthetic

pigments (Becker, 2004; Rommàn, et al., 2002). They are also potential therapeutic

agents in oxidative stress-induced diseases, and some authors have suggested that

biliproteins might also be effective in photodynamic therapy (He et al., 1996).

1.6 Thesis plan

This thesis is concerned with four different aspects of chromophore attachment to

phycobiliproteins.

1) The final step in phycobiliprotein biosynthesis is the covalent chromophore

attachment to the apoprotein. In vitro, spontaneous chromophore additions are

generally of low fidelity, therefore lyases were suggested that catalyze the

chromophore binding. However, at the beginning of my thesis such lyases were known

only for a sinlge binding site, cysteine-α84 of PC and PEC, the E/F type lyases. The

first goal was therefore to find and characterize phycobilin lyases which can attach

1. Introduction 15

phycobilin to other binding sites and other phycobiliproteins. This was triggered by a

meeting report (Shen 2004) that indicated a new class of lyases. While the groups of

Bryant and Schluchter worked mainly with Synechococcus PCC7002 that contains

only PCB chromophores, this work focused on Anabaena PCC7120 that has PCB and

PVB chromophores, and whose genome is available in the databases

(http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=BA000019)

2) Tooley et al. (2001) successfully biosynthesized α-PC in E. coli by using a

multi-plasmidic expression system. It is more advantageous than reconstitution in

vitro. Such a system seemed attractive, too, for searching and investigating new

layses, we therefore wanted to establish a multi-plasmidic expression system in E.

coli.

3) Currently, a variety of separation, purification and analytic methods are used to

analyze phycobiliproteins. Each method has a different application spectrum and

limitations, and can only explain some characters of phycobiliproteins. To fully

characterize the phycobiliproteins, a standardized multi-step protocol for separation,

purification and determination methods should be established. Since only few

reconstituted phycobiliproteins were fully characterized before the beginning of this

thesis, this analytical protocol should be applied to phycobiliproteins that were

reconstituted in E. coli both by the known E/F-type and the new lyases.

4) Even with the E/F-type lyases, there were very few mechanistic studies published.

A better characterization of the mechanism of new lyases will be done, using kinetic

studies, comparison of spontaneous and catalyzed attachment, and model studies

with modified substrate.

2. Materials and Methods 16

2: Materials and Methods

2.1 Materials

2.1.1 Organisms

E. coli strain:

BL21(DE3) (Studier et al.1986)

Cyanobacterium strain:

Fischerella sp. PCC7603 (Mastigocladus laminosus); Anabaena sp. PCC 7120 (Nostoc sp. PCC7120); Calothrix sp. PCC 7601; Spirulina platensis (Spary dried, Bonn, Gemany)

2.1.2 Vectors

pBluescript II KS (Stratagene, San Diego);

pET30a (Novagen, Germany);

Duet vectors (Novagen, Germany)

2.1.3 Plasmids

All plasmids were a generous gift of Prof. Kai-Hong Zhao (Huazhong University of

Science and Technology, China). They include three types: 1) lyase plasmids:

pCDFDuet-cpcS, pET-cpcS, pETDuet-cpcT, pCOLADuet-cpcE-cpcF,

pCOLADuet-pecE, pCDFDuet-pecF; 2) PCB biosynthetic plasmid:

pACYCDuet-ho1-pcyA; 3) apoprotein plasmids: pETDuet-pecA, pETDuet-cpcA,

pET-apcA1, pET-apcB, pET-apcA2, pET-apcD, pET-apcF, pET-cpcB,

pET-cpcB(C155I), pET-cpcB(C84S), pET-pecB, pET-pecB(C155I), pET-pecB(C84A).

The CPC and PEC genes come from M. laminosus, the other genes come from

Anabaena PCC 7120.


2.1.3 Chemicals

Generally, bidistilled water is used as well as chemicals of the highest obtainable

degree of purity unless indicated otherwise.

2.1.3.1 Buffers and solutions

The following table lists frequently used buffers and solutions (Table 2-1).

Table 2-1: list of frequently used buffers and solutions

Name Composition Coomassie-blue staining

Staining Solution: 0.15% (w/v) Coomassie Brilliant Blue G-250 (Serva) 40 % (v/v) Methanol 10 % (v/v) Acetic acid Destain Solution: 10 % (v/v) Methanol 10 % (v/v) Acetic acid

Di-potassium phosphate buffer (DPP-buffer)

50 mM K2HPO4 0.5 M NaCl pH 7.2

Fluka solution Fluka advanced protein assay (Sigma-Aldrich) in 1:5 dilution

with water IPTG 100 mM IPTG (Roth) in aqueous solution TB-Medium 1.2 % (w/v) Tryptone

2.4 % (w/v)Yeast extract 0.4% (v/v) Glycerol 17mM KH2PO4 72mM K2HPO4

LB-Medium 1 % (w/v) Tryptone

0.5 % (w/v)Yeast extract 1 % (w/v) NaCl pH 7.2 Sterilized by autoclaving for 20 min

RB- Medium 1 % (w/v) Tryptone


0.5 % (w/v) Yeast extract 0.5 % (w/v) NaCl 0.2 % (w/v) Glucose pH ranging from 6.8 to 7.5 Sterilized by autoclaving for 20 min

SDS-7 Marker Dalton Mark VII-L, MW range 14-70 kDa, Sigma SDS-PAGE loading buffer

625 mM Tris/HCl 1 M pH 6.8 2 % (w/v) SDS 10 % (v/v) Glycerine 0.003 % (w/v) Bromphenol blue 5 % (v/v) 2-Mercaptoethanol

SDS-PAGE running buffer

25 mM Tris/HCl pH 8.3 192 mM Glycin 0.1 % (w/v) SDS

SDS-Gel Stacking Gel 15% (for two chambers [8.3 x 7 x 0.075 cm])

1 ml Tris/HCl 3 M, pH 8.8 4 ml 30% Acrylamide/Bisacrylamide (35.7:1) 2.9 ml H2O 80 μl 10% SDS 40 μl 10% APS 4 μl TEMED

Separating Gel 5% (for two chambers [8.3 x 7 x 0.075 cm])

0.5 ml Tris/HCl 1 M, pH 6.8 0.65 ml 30% Acrylamide/Bisacrylamide (35.7:1) 2.8 ml H2O 40 μl SDS 20 μl APS 4 μl TEMED

Coomassie-blue staining solution

0.15% (w/v) Coomassie Brilliant Blue G-250 (Serva) 40 % (v/v) Methanol 10 % (v/v) Acetic acid

Coomassie-blue destaining solution

10 % (v/v) Methanol 10 % (v/v) Acetic acid

Zinc solution 1.5 M Zinc acetate FPLC-buffer

K2HPO4 50 mM NaCl 150 mM pH 7.0


Ni2+-solution 50 mM NiSO4 in aqueous solution Starting buffer for the Ni2+-chelating chromatography

20 mM K2HPO4, pH 7.0, 1 M NaCl

Washing buffer for the Ni2+-chelating chromatography

20 mM K2HPO4 pH 7.0 1 M NaCl 50 to 100 mM Imidazole

Elution buffer for the Ni2+-chelating chromatography

20 mM K2HPO4 pH 7.0 1 M NaCl 500 to 1000 mM Imidazole

EDTA buffer for the Ni2+-chelating chromatography

20 mM K2HPO4 pH 7.0 500 mM NaCl 50 mM EDTA

Reconstitution buffer for CpcS

K2HPO4 and KH2PO4 500 mM NaCl 100 mM pH 7.5

Reconstitution buffer for PecA

K2HPO4 50 mM NaCl 500 mM MnCl2 3 mM 2-Mercaptoethanol 1 mM Triton X-100 0,85% (v/v) Glycerin 10% (v/v) pH 7.0

Start buffer for Sep-Pak C18 column

0.1% (v/v)TFA

Elution solvent for Sep-Pak C18 column

Methanol

Start buffer for Bio-gel P-6 DG column

Dilute HCl pH 2

Elution buffer for Bio-gel P-6 DG column

30%(v/v) acetic acid in dilute HCl pH 2


2.1.3.2 Antibiotics

Antibiotic concentrations and related vectors are shown in Table 2-2

Table 2-2: Antibiotics and their used concentrations

Concentration (μg/ml)( E. coli)

Antibiotics Company Vectors Single

Plasmid Multiple Plasmid

Ampicillin Roth pBluescript; pETDuet 140 100 Chloramycetin Sigma-Aldrich pACYCDuet 17 10 Kanamycin Sigma-Aldrich pET30, pCOLADuet 30 20 Streptomycin Sigma-Aldrich pCDFDuet 25 15

2.1.4 Technical devices

Centrifuges

Refrigerated centrifuge:

Centricon H-401 (Kontron, Eching)

Sorvall RC-5B (Thermo, Waltham, MA, USA)

Rotor: F9S (Piramoon, Santa Clara, CA, USA)

Refrigerated benchtop centrifuges:

Sigma 3K18 C (Braun, Melsungen), Rotoren: 12154, 12155

Biofuge fresco (Kendro, Hanau)

Electrophoresis

Mini Protean III Dual Slab Cell (Bio-Rad, Munich Germany)

Constant Voltage Power Supply 1000/500 (Bio-Rad, Munich Germany)

UV/VIS-Spectrometer

UV-2401 PC Spectrometer (Shimadzu, Japan)

Lambda 25 Spectrometer (Perkin Elmer, USA)

Fluorescence Spectrometer

LS 55 Luminescence Spectrometer (Perkin Elmer, USA)


CD-Spectrometer

J-810 Spectropolarimeter (Jasco, Tokyo, Japan)

NMR-Spectrometer

DMX 750 Spectrometer (Bruker, Karlsruhe)

Mass Spectrometer

Micromass Q-Tof Premier (Waters Micromass Technologies, Manchester, UK)

Medium Nano-ES spray capillaries No. ES380 (Proxeon Biosystems, Denmark)

Chromatography for peptide purification

PD-10 (Sephadex G-25)-column (Amersham Biosciences, USA)

Sep-Pak C18 (Waters, USA)

Bio-gel P-6 DG (Bio-Rad, Munich Germany)

Vacuum concentrator

Eppendorf Concentrator 5301 (Eppendorf, Germany)

Fast Performance Liquid Chromatography

Series controller LKB GP-10, Pump P-50 (Pharmacia, Sweden)

Superdex 200 prep grade column 16/60 (Pharmacia, Sweden)

TIDAS UV/Vis Spectrometer and detector (J&M, Germany)

Software: Spectacle 1.55 (Lab Control, Germany)

High Performance Liquid Chromatography

Series controller 600 E multisolvent delivery system (Waters, USA)

TIDAS UV/Vis Spectrometer and detector (J&M, Germany)

Zorbax 300SB-C18 column (Agilent Technologies, USA)

Software: Spectacle 1.55 (Lab Control, Germany)

Documentation

Fluorescent gels: CCD KP 161 (Hitachi, GB)


Protein gels: hp scanjet 7400 c, reflection mode (HP, USA)

Others

Column matrix: Chelating SepharoseTM (Amersham Biosciences, USA) (affinity

chromatography)

Cold light source: Intralux 150 H universal (Volpi, USA )

Dialysis tubing: Servapor dialysis tubing 16mm (Serva, Germany)

Pump: Minipuls 2 (Gilson, Netherland)

Rotary evaporator: Rotavapor-R (Büchi, Germany)

Sonifier: Sonifier W-450 (Branson Ultraschall,Germany)

Shaker: Orbit Environ Shaker (Lab-Line, USA)

Data analysis

Software employed for advanced data processing and visualization includes:

Data transfer: Spectacle 1.55 (Lab Control, Germany)

General analysis: Origin 7.0 (Microcal Software, USA), Microsoft Excel 2003 (Microcal

Software, USA), Adobe Photoshop 6.0 (Adobe Photoshop, USA)

Kinetic analysis: SPECFIT/32 (Spectrum Software Associates, USA)

Analysis of NMR spectra: MestReC (Mestrelab Research SL, Spain)

Word processing: Microsoft Word 2003 (Microcal Software, USA)

2.2 Methods

2.2.1 General molecular biological methods

General molecular biological methods such as growing conditions of bacteria,

restriction endonuclease digestion, DNA ligation, agarose gel electrophoresis,

transformation of E. coli and recombinant DNA procedures were carried out according

to standard procedures as described in Sambrook et al., (1989). Plasmid DNA

isolation, PCR and gel purification were prepared using Qiagen plasmid mini kits

12125 (Hilden, Germany) according to manufacturer’s protocols.

2.2.2 Protein isolation


For convenient preparation of proteins, a heterologous system was established by

Zhao et al. (2002), using the expression vector pET-30a (Novagen, Schwalbach) with

intrinsic kanamycin-resistance and D-galactose-inducible promoter. Additionally,

His6-tagged derivatives were constructed to allow for easy purification of the gene

products.

2.2.2.1 Protein expression in Escherichia coli

The pET-based plasmids were used to transform E. coli BL21(DE3). Cells were grown

in Luria-Bertani (LB) medium containing kanamycin (see Table 2-2 for antibiotic

concentrations) at 37 °C for genes from M. laminosus or at 20 °C for genes from

Anabaena sp. PCC 7120. For expression of untagged proteins, which is available with

the Duet vectors (Novagen, Germany), kanamycin has to be substituted by other

antibiotics in different final concentrations (see Table 2-2).

Once the cultures have reached the exponential growth phase (after 4-5 hr, A600 =

0.5–0.7), expression of the foreign gene is induced with IPTG (1mM). Cells were

collected by centrifugation (7000 xg, 15min) 5 hr after induction (for genes from M.

laminosus) or after 6 hr (for genes from Anabaena sp.), then washed twice with

doubly distilled water and stored at -20 °C until use.

2.2.2.2 Protein purification

Cell pellets were resuspended in ice-cold start buffer and disrupted by sonication (30

min at 150 W, duty cycle 40%). The suspension was centrifuged at 12,000 xg for 30

min at 4 °C. The crude protein-enriched supernatant was stored at 4 °C until use.

Ni2+-affinity chromatography

Subsequently, the supernatant was purified via Ni2+-affinity chromatography on

chelating Sepharose (Amersham Biosciences). The His6-tag coordinates the bivalent

cation and is adsorbed to the column, while the major fraction of the bacterial proteins

has generally no or little affinity for the column material. 30 ml of the supernatant were

applied to a Ni2+- chelating column (60 x 20 mm) equilibrated with start buffer (see Tab.

2-1 for all solutions and buffers), and then were washed with start buffer and elution

buffer. The following washing steps increase in stringency to enhance the purification


process. To finally elute the product fraction, high imidazole concentration (0.5-1M) is

applied, which is a strong chelator of Ni2+ ions, thus displacing the histidines and

releasing them from the stationary phase.

After collection, the protein fractions were dialyzed twice against the initial buffer and

kept at -20 °C until use.

For regeneration of the column, EDTA buffer is used to strip off Ni2+ and protein

remainders. After excessive rinsing with water, Ni2+-solution is applied to recharge the

column. Consecutive equilibration with starting buffer prepares the column for

application of another protein sample. In case the EDTA buffer is not sufficient to

clean the column (when a “dirt veil” is still visible after the rinsing step), a washing step

with 0.5 M NaOH can be included for improved cleaning. If not in use, the column is

stored in 20% ethanol.

Fast Performance Liquid Chromatography

Samples (6 ml) in Tris buffer (1 M, pH 6.0) were concentrated to an overall volume of

1 ml using centrifugal filter devices (Amicon Ultra-15, Millipore, USA). They were then

applied to the gel column, Superdex 200-16/60 preparative grade (Amersham

Biosciences, flow rate 0.75 ml/min) equilibrated with Tris buffer (1 M, pH 6.0). The

eluant was monitored with a diode array detector (Tidas, Diode-Array-Photometer,

J&M, Germany; 280–800 nm, measurement interval 5 s). Fractions were collected

and analyzed by SDS-PAGE according to Laemmli (1970) using 15% stacking and

5% separating gels (Boehm, 2006).

2.2.2.3 Protein analysis

After dialysis, the solutions are retrieved, transferred to Falcon tubes, and their protein

contents were evaluated employing two methods:

Sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gels

50 μl of the protein solution are mixed with 50 μl SDS-PAGE loading buffer and boiled

for 5 minutes. 10-20 μl of the sample are applied to a two step SDS-PAGE gel (see

table 2-1) and run for 30 min of 50 V, followed by 75 min of 150 V. The gel is

developed by overnight incubation in staining solution, and subsequently excess dyes

removed by destaining solution, until the desired degree of contrast is achieved.


Chromophorylated protein species are identified by Zn2+-induced fluorescence prior to

Coomassie staining: after soaking the gels in 1 M Zn2+ acetate solution for 15 min,

UV-light (360 nm) induced fluorescence can be observed (Berkelman and Lagarias,

1986).

Protein assay

Protein concentrations were determined by protein assay kits from Fluka advance

(Seelze, Germany).10 μl of the protein solution are mixed with 300 μl FLUKA solution

and 690 μl distilled water, and incubated for several minutes. Absorption of the protein

sample is measured at 590 nm and the concentration in mg/ml deduced by

comparison to an albumin standard. For protein solutions in the expected

concentration range of 5-10 mg/ml, as in this case, a 1:10 dilution with distilled water

proved to give convenient absorption values. Comparing this value with the

corresponding lanes on the SDS-PAGE and taking into account the contamination

with co-purified E. coli proteins, a rough estimate of the concentration of the products

is possible. Since the molar masses for the proteins are known, the concentration in

mg/ml can be converted to μmol/ml. 1 L expression medium usually yields 10 ml

protein solution (5-10 mg/ml in concentration as determined by the FLUKA Protein

assay) which are equivalent to 4-6 μmol protein.

2.2.2.4 Concentration of proteins

In case the volume of a protein solution needs to be reduced to increase its relative

protein concentration, it is transferred to a centrifuge filter (Biomax 10K 15 ml,

Millipore) and centrifuged (4 °C, 7 000 xg) until the desired volume reduction is

achieved.

2.2.2.5 Protein storage

The protein solutions are stable at room temperature down to 4°C for approximately 2

days. Long-term storage is possible at -20 °C but accompanied by loss of specific

enzyme activity.

2.2.3 Pigment isolation

Pigments are light-sensitive and prone to oxidation. All work that involves pigments is


therefore performed under green protective light or at least dim light. During

phycocyanobilin (PCB) extraction, when the pigment-containing solutions are heated,

argon is employed as protective gas to prevent excessive exposure to oxygen,

thereby reducing the formation of oxidation- and breakdown products.

2.2.3.1 Pigment extraction from Spirulina platensis

Spray-dried cells of Spirulina platensis are methanolyzed and free pigment is

extracted with chloroform. Crude pigment is obtained after solvent evaporation. The

detailed procedure has been described previously (Storf et al., 2001).

2.2.3.2 Phycocyanobilin purification

Chromatography of the PCB crude extract on a reversed phase column separates

pure PCB from its breakdown products, derivatives, and remainders of other pigments.

Stationary phase: Silica gel RP 8 (23-63 μm) 60 Å (ICN, Eschwege); mobile phase:

40% 2-Propanol, 60% 50 mM KPP buffer pH 2.1 (v/v) (degassed). Pressure is applied

to increase the flow; the column is run under protective light conditions. The dark blue

fraction is collected, and the solvent evaporated. For details see Storf et al. (2001).

2.2.3.3 Phycocyanobilin analysis

The pigment thus obtained is redissolved in methanol. 5 μl are diluted in 1 ml

methanol, acidified with concentrated HCl (2% v/v), and a spectrum is recorded in the

range from 300-800 nm. From the known extinction coefficient ε690 = 37 900 M-1cm-1

(Cole et al., 1967) of PCB in acidic methanol, the concentration of the pigment

solution can be deduced. The position of the red maximum indicates the degree of

pigment purity: Pure PCB peaks at 690 nm, while contamination with breakdown

products or derivatives causes a blue shift. For reconstitution assays, PCB is

dissolved in DMSO instead of methanol.

2.2.3.4 Phycocyanobilin storage

The pigment is dissolved in little methanol, aliquots are transferred to small vials, and


the solvent is evaporated with a gentle stream of argon. The vials are closed tightly

and can be stored for extended times at -20 °C under argon atmosphere.

2.2.4 Phycobiliprotein reconstitution/preparation

For convenient preparation of the phycobiliproteins, a multiple-plasmid expression

system was established by Zhao et al. (2006a) using the expression vector Duet

(Novagen, Germany).

2.2.4.1 Phycobiliprotein expression

One of the apo-phycobiliprotein plasmids, one of lyase plasmids and

pACYCDuet-ho1-pcyA were transformed together into BL21 (DE3) cells to produce

phycobiliprotein. Cells were grown at 37 °C in LB-medium under the respective

antibiotic selections. When the cell density reached A600=0.5–0.7, IPTG (1 mM) was

added, cultivation continued for 12 hr at 20 °C in darkness. Cells were then collected

by centrifugation (7000 xg, 15min), washed twice with doubly distilled water, and

stored at -20 °C until use.

2.2.4.2 Biliprotein purification

see 2.2.2.2

2.2.4.3 Biliprotein extinction coefficient

The extinction coefficients of the reconstituted and biosynthesized chromobiliproteins

were determined based on the extinction coefficient of PCB in CPC in 8 M acidic urea

(pH=1,5) (ε660=35,500 M-1cm-1) (Glazer and Fang ,1973); and PVB in α-PEC in 8 M

acidic urea (ε590=38,600 M-1cm-1 ) (Bishop et al. , 1987).

2.2.4.4 Biliprotein fluorescence yields

Fluorescence yields ΦF of the reconstituted and biosynthesized chromobiliprotein

were determined in KPP buffer (20mM, pH 7.2), using the known fluorescence


quantum yield (ΦF = 0.27) of CPC from Anabaena sp. PCC 7120 (Cai et al., 2001) as

standard.

2.2.4.5 Chromoprotein digestion method

Reconstituted or biosynthesized chromoproteins were purified by Ni2+

chromatography (see 2.2.4.2) and dialyzed against KPP (20mM, pH 7.2). For pepsin

digestions, the desired chromoprotein solution was acidified with HCl to pH 1.5, and

pepsin was added to the chromoprotein (1:1, w/w). For trypsin digestions, the dialyzed

chromoproteins (10 μM) were digested with trypsin (40 μM) in KPB (100 mM, pH 7.0)

In both cases, the mixture was incubated for 3 hr or 4hr at 37 °C, and then

fractionated on Bio-Gel P-6 DG (Bio-Rad) equilibrated with dilute HCl (pH 2.5).

Colorless peptides and salts were eluted with the same solvent, and the adsorbed

chromopeptides were eluted with acetic acid (30%, v/v) in dilute HCl (pH 2.5). The

collected samples were desalted and concentrated by Sep-Pak C18 (Waters, USA),

then eluted with 100% methanol to small vials, and the solvent is evaporated with a

gentle stream of argon until the desired volume. The vials are closed tightly and can

be stored for extended times at -20 °C under argon atmosphere until use.

2.2.4.6 Semipreparative HPLC

The enzyme digested samples were fractionated by repeated HPLC. The collected

samples were dried with a Vacuum concentrator (45 °C), and stored at -20 °C until

use.

The first preparative program

Solvent: A: KPP (100 mM, pH 2.1) B: acetonitrile

Gradient:

time (min) 0 2 22 25 45

A:B 80:20 80:20 60:40 80:20 80:20


Flow rate: 1.0 ml/min

The second preparative program:

Solvent: A: formic acid (0.1%, pH 2.0) B: acetonitrile containing 0.1% formic acid

Gradient:

time (min) 0 2 32 35 55

A:B 80:20 80:20 60:40 80:20 80:20

Flow rate: 1.0 ml/min

All solvents applied to the column are degassed and filtered through a sterile 0.22

μm-membrane. When preparations are finished, the column is equilibrated with

acetonitrile for half hour for better preservation.

2.2.5 Lyase CpcS (Alr0617) reaction assay

Chromophore reconstitution with apo-phycobiliproteins was assayed as described

before (Zhao et al. 2006a, 2007), using the following standard reaction conditions:

potassium phosphate buffer (KPB, 500 mM, pH 7.5) containing NaCl (100–150 mM),

CpcS (10–30 μM), and apo-phycobiliprotein (10–30 μM). PCB (final concentration =

5–10 μM) was added to the proteins as a concentrated dimethylsulfoxide solution, the

final concentration of dimethylsulfoxide did not exceed 1% (v/v). The mixtures were

incubated at 35 °C for 0.5–4 hrs in darkness.

2.2.6 Chromophore adduct assay

2.2.6.1 Reconstitution system

PCB and 15Za-PCB were reconstituted with imidazole (50 - 500 mM ) or ME

(10-600mM), and/or CpcS (10 - 50 μM) by incubation in KPP (500 mM, pH 7.5) for

30min-2hr.


DOHH was incubated with PecA, PCB or/and PecE, or/and PecF in holo-PecA

reconstitution system.

2.2.6.2 Product purification and digestion methods

See 2.2.2.2 , 2.2.4.5 and 2.2.4.6

2.2.6.3 Analytical methods

The chromophore adducts were analyzed spectroscopically (absorption, fluorescence,

circular dichroism) and by SDS-PAGE using Zn2+ staining (see 2.2.2.3). The HPLC

products were analyzed mass spectrometry and NMR spectrometry.

2.2.6.4 Transfer assay

The purified chromophore adduct was mixed with the respective apo-phycobiliprotein

(10–30 μM)and lyase(10-30 μM), and the reaction followed at 35 or 20°C for 1–3 hr by

absorption and/or fluorescence spectroscopy.

2.2.7 Spectroscopy

2.2.7.1 UV/Vis spectroscopy

UV visible absorption spectra at room temperature were measured on Shimadzu

UV-2401PC or Perkin-Elmer Lambda 25 spectrophotometer using 1 cm or 1 mm path

length cuvettes. Formation of the photochromic PVB-PecA (i.e. α-PEC) in the lyase

reaction was monitored by the absorption at 570 nm and by double-difference

spectroscopy of the reversible photoreaction of the PVB chromophore, as previously

described (Zhao et al. 1995).

2. 2.7.2 Fluorescence spectroscopy

The fluorescence spectra of chromoproteins were recorded by a LS 55 Luminescence

Spectrometer (Perkin Elmer, USA) using 1 cm path length cuvette at room temperature.

Excitations were scanned from 300- 850 nm with the emission wavelength set to the

desired wavelength in the range 500 - 660 nm.


2.2.7.3 Electrospray ionisation mass spectroscopy (ESI-MS)

The isolated colored products were dissolved in spray solution (65 % methanol, 35 %

water, 0.1 % formic acid ), and analyzed by mass spectrometry in positive ion mode

using a Micromass Q-Tof Premier mass spectrometer (WatersMicromass

Technologies, Manchester, U.K.) with a nano-ESI source. Work was done in

collaboration with Dr. M. Plöscher and Prof. Dr. L. A. Eichacker (Munich University,

Germany).

2.2.7.4 Circular dichroism (CD) spectroscopy

Circular dichroism measurements were performed with a J-810 CD spectropolarimeter

(JASCO Corp., Japan) in 1 mm (for far-UV region) or 1 cm (for visible light region)

rectangular Quartz cuvettes at 22 °C.

2.2.7.5 Nuclear magnetic resonance (NMR) spectroscopy

The isolated colored products were dissolved in 0.1% TFA/D2O, and transferred with

thinly drawn-out Pasteur pipettes to 2 mm diameter NMR microtubes. NMR spectra

were measured in collaboration with Dr. R. Haessner (Munich Technical University,

Germany) at 281 K (Bruker DMX601 operating at 600.14 MHz).

2.2.8 Websites

Protein sequences were taken from the sequences of database Swiss-Prot, TrEMBL

(http://www.expasy.org) and of the National Centre for Biotechnology Information

(NCBI) (http://www.ncbi.nlm.nih.gov). Sequences alignments were done by using

BLAST (http://www.expasy.org/tools/blast/) or CLUSTAL W (http://www.ebi.ac.uk/

clustalw).

3. Results and Discussion 32

3: Results and Discussion

3.1 Structure analysis of reconstituted holo-PecA

3.1.1 Reconstitution principle of holo-PecA

Phycocyanobilin(PCB) is biosynthesized from heme by the action of two enzymes,

heme oxygenase and BV-reductase. PCB is then attached to the apoprotein by a

heterodimeric lyase, PecE/PecF, that concomitantly isomerizes the chromophore to

phycoviolobilin(PVB) (Zhao et al. 2000; Storf et al. 2001). Using endogenous heme as

substrate, Tooley et al.(2002) introduced five genes into E. coli to produce

holo-HT-PecA from Anabaena PCC7120 with optical spectroscopic properties

characteristic of the native holo subunit. Besides the genes for the apoprotein (pecA)

and the lyase (pecE/F), these included the two respective genes for PCB synthesis,

viz. ho1 and pcyA, from the same organism (Fig.3-1).

It is known that in vitro tests of lyase function are hampered by the spontaneous and

faulty addition of chromophores. Since there was evidence that this activity is

suppressed in the E. coli system, a multi-plasmidic system was established in our lab,

too, to generate holo-biliproteins in E. coli. This also served a more thorough analysis

of the products, including mass, NMR and CD spectroscopy.


NH

N HN

COOHCOOH

HN

OOH

A

BC

D

1 2

34

5

6

1819

17

16

15

14

31

7

89

1011

12

13

Cys

SH

NN N

COO-COO-

N O

SCys

H

H

H

OH

H

Apo-α -phycoerythrocyanin 3Z-phycocyanobilin

Biliverdin αⅨ

NH

N N

COOHCOOH

HN

OO

H

D

C B

A

Heme HO1

+

PecE+PecF

Holo-α -phycoerythrocyanin

PcyA

84

84

FIG. 3-1: Biosynthetic pathway of PCB from heme and its addition to the phycoerythrocyanin apo-α subunit and isomerization to PVB.

3.1.2 Expression and Purification of the holo-PecA

Expressing the pETDuet-pecA, pCOLADuet-pecE, pCDFDuet-pecF and

pACYCDuet-ho1-pcyA plasmids in the strain E. coli strain BL21(DE3), upon induction

with IPTG, the culture acquired a pronounced pink tint. The His-tagged holoprotein

(holo-HT-PecA) was purified by immobilized Ni2+ affinity chromatography, and its

spectroscopic properties were determined with the protein fraction purified by

immobilized metal affinity chromatography after dialysis against 50 mM K phosphate

(pH 7.0). The absorbance spectrum of holo-HT-PecA had a λmax at 561 nm and the

fluorescence had a λmax at 582 nm (Fig. 3-2). The corresponding values for in vivo


reconstituted holo-PecA from Anabaena PCC7120 are 561 nm and 582 nm (Tooley

and Glazer, 2002). The spectrum corresponded closely to that of the native holo-PecA

obtained from Anabaena PCC7120 (Bryant et al., 1976).

0

100

200

300

400

500

fluor

esce

nce

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abso

rptio

n [a

.u.]

Wavelength [nm]

FIG.3-2. Spectroscopic properties of Anabaena PCC7120 holo-PecA expressed in E. coli. Shown are the absorption (solid) (λmax = 561 nm) and fluorescence emission spectra (dashed) (λmax =582 nm for holo-PecA purified from E. coli by affinity chromatography.

Analysis of Coomassie brilliant blue-stained gels after SDS-PAGE of the purified

protein showed that there were three distinct bands (Fig. 3-3, lane A), band 1 at ~28

kDa, band 2 at ~20 kDa, and band 3 at ~19 kDa. The sizes of these bands are

compatible with those calculated for PecE, holo-HTPecA, and PecF, respectively.

This result is like that of Tooley et al. (2002). Upon exposure to Zn2+ (Berkelman et al.,

1986) and UV illumination, only band 2 was fluorescent, indicating that it contained

covalently attached bilin (Fig.3-3, lane a).

FIG. 3-3: SDS-PAGE analysis of Anabaena PCC7120 His-tagged proteins expressed and purified by affinity chromatography. Proteins were visualized by Coomassie brilliant blue staining (left panel) and by the UV excited fluorescence of bilin-bearing polypeptides in the presence of Zn2+ (right panel). Lanes A, a:PecE, HT-PecA, and PecF (bands 1, 2, and 3, respectively) expressed in E. coli strain; lanes B, b: molecular mass standards (from the top, 45, 36, 29, 24, 20 and 14 kDa).

A B a b

123


The extinction coefficient ε563=99000 M-1cm-1 of in vivo reconstituted holo-PecA was

similar to that for native Z-PecA. obtained from Anabaena PCC7120 (ε568=96000

M-1cm-1) (Siebzehnrübl, 1990), and higher than for holo-HT-PecA produced in vitro by

the addition of PCB to a mixture of HT-PecA, PecE, and PecF in 100 mM phosphate

buffer at pH 7.0 ( ε567=77000 M-1cm-1)( Storf et al.,2001). A fluorescence yield ΦF =

0.22 of in vivo reconstituted holo-PecA was measured.

3.1.3 Circular Dichroism Spectroscopy

The CD spectrum of biosynthesized holo-PecA was similar to that of native α-PEC

from M. laminosus (Parbel et al.1997; Wiegand et al. 2002), that is, with a strong

positive Vis band at 563nm (Parbel: 563nm; Wiegand: 566nm) and a negative

near-UV band at 330nm (Parbel: 330nm; Wiegand: 329nm) (Fig.3-4), these signals

are typical for α-PEC in the Z-configuration. The far-UV CD spectrum is typical for

α-helical proteins.

300 400 500 600 700 800-20

-15

-10

-5

0

5

10

15

20

25

CD

[a.u

.]

Wavelength [nm]

200 220 240 260 280 300

-10

-8

-6

-4

-2

0

2

4

6

CD

[a.u

.]

Wavelength [nm]

FIG. 3-4: CD spectroscopy of reconstituted holo-PecA Vis- CD (top) and far-UV CD spectrum (bottom) of Anabaena PCC7120 holo-PecA reconstituted in E. coli purified by immobilized Ni2+ affinity chromatography.


3.1.4 Photoisomerization of α-PEC

Its responsiveness to light of certain wavelengths makes α-PEC, the youngest

member of the phycobilin protein family (Apt et al., 1995), unique among the

phycobiliprotein subunits. The isolated PEC α-subunit is photoreversibly

photochromic (Björn, 1979), reminiscent of plant phytochromes, but with sensitivity to

orange/green instead of red/far-red irradiation (Kufer and Björn, 1989). Light of

appropriate energy content leads to the spectral changes in α-PEC illustrated below.

After irradiation with 570 nm light, the band at 565 nm decreases and a new

absorption band arises at 505 nm. This effect is fully reversible when the sample is

irradiated with 500 nm light. Subtraction of the 500 nm spectrum from the 570 nm

spectrum gives a difference spectrum with a characteristic shape for a band shift

related to the photoisomerization event, as seen in Fig. 3-5 A and B.

300 400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

Z conformation E conformationA

300 400 500 600 700 800-0.6

-0.4

-0.2

0.0

0.2

0.4

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

B

A565max

ΔA

FIG. 3-5: Photoisomerization of α-PEC A: Absorption spectra of α-PEC (of Anabaena sp. PCC 7120, in vivo reconstituted and affinity purified) after irradiation with 500 nm light (solid line) and 570 nm light (dotted line). B: Difference spectrum (570 minus 500 nm) of the same sample with the characteristic maximum at ~500 nm and minimum at ~570 nm

The photoactivity of reconstituted holo-PecA from Anabeana PCC7120(ΔAA= 79%) is

smaller than that of native holo-PecA (ΔAA=101%) from Mastigocladus laminosus

(Zhao et al., 1995). This may be due to slightly different spectra of the two species.


The molecular changes induced by irradiation with 570/500nm light are divers. In the

chromophore, it has been assigned to a E/Z-isomerization of the Δ15,16 double bond

between the C and D pyrrole rings (Zhao et al., 1995; Schmidt et al., 2006; Duerring et

al. 1990; see Fig.3-6), in which the E-isomer has a strongly twisted conformation at

this position.

FIG. 3-6: E-Z isomerization of PVB in the PEC α-subunit left: E-constitution of PVB as induced by 570 nm light, right: Z-constitution of PVB as induced by 500 nm light

In the dark, either of these states is quite stable. The 500 nm absorbing E-form shows

little fluorescence, it is higher in energy, but transfer to other biliproteins occurs

efficiently only from the fluorescent Z- form absorbing at 570 nm (Storf, 2003).

In native α-PEC, PVB is predominantly present in the 15Z configuration, and the SH

groups are reduced (Parbel et al., 1997). However, the presence of a distinct shoulder

near 565 nm in the E-spectrum and of a less defined shoulder near 505 nm in the Z

-spectrum (compare FIG. 3-5) give evidence that the transformation is incomplete,

due to band overlap. This is true for the reconstituted as well as the native sample.

3.1.5 HPLC–ESI-MS analysis of chromopeptides from holo-PecA

RP-HPLC separation of trypsin digest (Fig.3-7, top) on a C18 column gave two main

colored components. Component 1 (tr=13.3 min) had an absorption maximum at 592

nm that is typical for a PVB-containing peptide, component 2 (tr=28.4 min)

corresponds to free PCB according to its retention time and the absorption maximum

at 686 nm (Fig.3-7). The fraction with retention time 13.3 min was used for MS.


300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavenlength [nm]

tr=13.3min tr=28.4min

FIG. 3-7: HPLC and absorption of the chromopeptide obtained from tryptic digestion of the reconstituted holo-PecA.. HPLC was eluted using a gradient (80:20 to 60:40) of formic acid (0.1%, pH 2) and acetonitrile containing 0.1% formic acid.

The sequence of the chromophore-containing peptide was deduced from sequence

around the chromophore-binding sites in holo-PecA from Anabaena PCC7120. PVB

is bound to cysteine resides 84 of the alpha subunit. (http://www.ncbi.nlm.nih.gov/

entrez/viewer.fcgi?db=protein&id=17129869). There is excellent agreement between

the measured peptide Mr values and peptide Mr values calculated from sequences of

alpha subunits of holo-PEC from Anabaena PCC7120 (Table 3-1). Sequences of

these peptides could be found when MS–MS capabilities of the instrument are


employed. That will make this method comparable in performance to amino acid

sequencing of chromophore-containing peptides (Klotz and Glazer, 1985). The

chromopeptide sequence C(PCB)VR confirms covalent chromophore binding to

Cys-84 of the holo-PecA apo-protein.

. Table 3-1 Mass of chromopeptide(component 1) from tryptic digestion of holo-PecA

In the case of pepsin digestion, separations were equally efficient (Fig.3-8). Trypsin

cuts amino-acid chains on the C terminus of lysine and arginine. Pepsin is relatively

non-specific but preferentially cleaves the peptide bonds of hydrophobic amino acids.

Triply charged ions found on peptides from trypsin digest originate from positive

charges on the N terminus of the peptide, the N terminus of arginine, and the

positively charged chromophore. Doubly charged ions from pepsin digest originate

from the positively charged peptide N terminus and the positively charged

chromophore (Isailovic et al. 2004). However, mass spectroscopy of tryptic

chromopeptides was consistently better than that of peptic chromopeptides, therefore

always tryptic digests were used for MS. By contrast, NMR spectra turned out to be

consistently more reliable and cleaner with peptic peptides, possibly because the

chromophore is more stable under acidic than at neutral conditions. So I choose

tryptic chromopeptides to measure MS and peptic chromopeptides to measure NMR.

3.1.6 NMR analysis of peptic chromopeptides from holo-PecA

RP-HPLC separation of pepsin digest (Fig.3-8) was achieved on a C18 column.

Chromopeptides having retention times of 3.0 and 6.6 min show absorption maxima

589 nm and 591nm, respectively (inset of Fig.3-8), and correspond to PVB-containing

peptides. The fraction with retention time 3.0 min was used for NMR spectroscopy.

Peptide mass chromopeptides Chromopeptide sequence Calc. Expt.

PecA-PVB C(PVB)VR [M+2H]2+ 481.38 481.34


0 10 200.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Abs

orpt

ion

[a.u

.]

Retention time [min]

1

2

300 400 500 600 700 800

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

FIG. 3-8: HPLC(λdetect=590nm) and absorption spectra (insert) of the chromopeptides obtained from peptic digestion of the reconstituted holo-PecA. HPLC conditions as in Fig. 3-7. Absorption spectra of peak 1 (solid line, λmax=589 nm) and of the peak 2 (dashed line λmax=591 nm).

The 1H NMR spectra of chromopeptide (tr=3.0) from holo-PecA of Anabaena PCC

7120, in vivo reconstituted and pepsin digested, were measured. All assignments are

made by using the A-ring linkage numbering scheme (see Fig 3-1) and are given in

Table 3-2. The full 1-D 1H NMR spectrum is shown in Fig. 3-9.

8 7 6 5 4 3 2 1 0

Chemical shift [ppm]

HDO

FIG. 3-9: 1-D H-NMR spectrum of PecA chromopeptide in 10 mM TFA/D2O at 25℃


Table 3-2: 1H NMR(600-MHZ) Assignments for peptide SKC(PVB)VR of reconstituted PecA-PVB (10mM TFA/D2O)

Chemical shift Multiplicity (JH-H, [Hz])

Number of H’s Assignment

7.51 s 1 10-H 6.25 s 1 15-H 4.55 dd (5.94,6.79) 1 4-H 4.07 m 1 3’-H 3.67 d (5.66) 1 5-H 3.03 d (7.00) 4 8,12-CH2CH2COOH 2.84 m 4 8,12-CH2CH2COOH 2.48 m 1 5-H 2.32 m 2 18- CH2CH3 2.09 s 2.00 s 1.98 s

12 2,7,13,17-CH3

1.29 d ( 6.98) 3 3’- CH3 0.91 m 3 18- CH2CH3 4.49 m 1 Cys α- CH 3.07 m 2 Cys β- CH2 4.19 d ( 6.96) 1 Val α- CH 1.90 m 1 Val β- CH 0.83 d ( 6.41) 6 Val γ- CH3 4.21 d ( 9.05) 1 Arg α- CH 1.53 m 4 Arg β,γ- CH2 2.90 m 2 Arg δ- CH2 4.41 m 1 Ser α- CH 3.78 m 2 Ser β- CH2 4.36 t (5.57) 1 Lys α- CH 1.48 d (7.13) 4 Lys β,γ- CH2

Analyzing the downfield region of the spectrum, one observes the two vinylic 10-H

and 15-H resonances at 7.51 and 6.25 ppm, respectively. These peaks correspond

well to the 10-H and 15-H singlets previously observed in A-ring-linked peptide α-1

PXB from M. laminosus (Bishop et al., 1987). Noticeably absent, however, is a

downfield vinylic resonance corresponding to 5-H. The β-1 PCB chromopeptide of

C-phycocyanin exhibits this proton as a singlet at 5.94 ppm (Lagarias et al., 1979).

PVB lacks the methine bridge between rings A and B, therefore, shows no such

resonance, the lack of the respective signal is taken as proof of saturation between

the A and B rings. Because the mass spectral data prove the isomeric relationships of

PVB and PCB, this double bond must exist elsewhere on the PVB-tetrapyrrole moiety.


Previous studies showed that the PEB and PUB chromophores have an exocyclic

vinyl group attached at C-18 (Klotz et al., 1985; Nagy et al., 1985), typically

manifested as a downfield pattern in the 1H NMR spectrum. No such group is

observed in the spectrum of PVB. The only alternative position for this unsaturation is

between C-2 and C-3, which is corroborated by the 1H NMR spectrum. Although only

3 vinylic methyl singlets are seen between 1.98 and 2.09 ppm, integration establishes

a total of 12 protons; therefore, 4 vinylic methyl groups (C-2, C-7, C-13, and C-17) are

actually present, thereby proving the presence of a double bond between C-2 and

C-3.

The broad doublet of doublets at 4.55 ppm is assigned to 4-H, and coupling with the

two nonequivalent 5-H protons at 3.67 and 2.48 ppm is obvious. The doublet at 1.29

ppm is assigned to 31-CH3 and a multiplet at 4.07 ppm is assigned 31-H. The

resonances from the propionyl side chains are found at 3.03 and 2.84 ppm. The ethyl

group is seen as two multiplet at 2.32 ppm (18- CH2CH3) and 0.91ppm (18-CH2CH3).

All PVB NMR resonances are therefore similar to those in the chromopeptide of α-1

PXB from M. laminosus (Bishop et al., 1987), the chromophore has since been

renamed PVB.

At the same, the resonances from the amino acids Ser, Lys, Cys, Val, Arg are found in

the NMR spectrum. Sequences of chromophore-containing peptides were deduced

from sequences around the chromophore-binding sites in holo-PecA from Anabaena

PCC7120. The chromopeptide is SKC(PVB)VR.

3.1.7 Discussion

The studies described here documented successful reconstitution of the predicted

pathway for the biosynthesis of the phycoerythrocyanin holo-α-subunit (Fig.3-1) in E.

coli. The results of experiments also support the conclusions reached by Tooley et

al.(2002, in vivo), Zhao et al. (2000, in vitro) and Storf et al. (2001, in vitro) that the

heterodimeric lyase/isomerase (PecE/PecF) catalyzes both the covalent attachment

of PCB and the concurrent isomerization of the molecule to PVB.

Jung et al. (1995) established that in Anabaena PCC7120 interposon mutagenesis of

pecE, pecF, or pecE and pecF eliminates the formation of holo-PecA. They showed


that ~30% of the wild-type level of PecB was present in the phycobilisomes of all

these mutants. In contrast, holo-PecA was barely detectable in the pecE and pecF

mutants, but it was present in a modified form in the pecEF deletion mutant in a 1:1

ratio with the PecB. Instead of PVB, the chromoprotein contained PCB, as identified

by isolation of the subunit and amino-terminal sequencing.

PecA forms a complex with both lyase subunits, PecE and PecF (in section 3.1.2).

The identities of all three components in the holo-HT-PecA–PecE–PecF ternary

complexes were established unambiguously by protein and tryptic peptide analyses

performed by matrix-assisted laser desorption ionization–time of flight mass

spectrometry (Tooley et al. 2002). Probably, complex formation of in the single

subunit mutants with the respective complementary subunit prevents addition of PCB,

possibly by another lyase. This inhibition would be relieved in the double mutant.

PecA and PecE form a weak complex that is stabilized by PCB. It has been

suggested that the first reaction step involves a conformational change and/or

protonation of PCB, and that PecE has a chaperone-like function on the

chromoprotein (Boehm et al. 2007).

Zhao et al. (2002) studied the cofactor requirements and enzyme kinetics of the novel,

dual-action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin-α84-

cystein-lyase (PVB-PEC-lyase) from M. laminosus, which catalyses both the covalent

attachment of phycocyanobilin to PecA, the apo-α-subunit of phycoerythrocyanin, and

its isomerization to phycoviolobilin. Mercaptoethanol and the divalent metals, Mg2+ or

Mn2+, were required, and the reaction was aided by the detergent, Triton X-100.

Phosphate buffer inhibits precipitation of the proteins present in the reconstitution

mixture, but at the same time binds the required metal. Kinetic constants were

obtained for both substrates, the chromophore (Km =12–16 μM, depending on [PecA],

kcat = 1.2•10-4 s-1) and the apoprotein (Km = 2.4 μM at 14 μM PCB, kcat =0.8•10-4 s-1).

A multi-plasmid system was established for reconstitution of holo-PecA in E. coli.

HPLC, FPLC, affinity chromatography and SDS–PAGE separations followed by

absorbance, fluorescence, circular dichroism, MS and NMR detections were used for

spectroscopic and structural characterization of reconstitution product or the

chromopeptides obtained from enzymatic digestion. This also established a

methodology to analyze of other phycobiliproteins and fluorescent proteins in general.


After cloning of apophycobiliprotein genes, phycobilin biosynthesis genes and lyase

genes from several cyanobacteria, various phycobiliproteins could be

biosynthesized in the heterologous E. coli system using dual plasmids containing

their respective genes. BL21(DE3) cells could be transformed to express up to eight

proteins in the desired combination. This heterologous system in E. coli is more

advantageous than reconstitution in vitro in the following two respects: (1) On

reconstitution in vitro, where the respective genes were first overproduct in E. coli,

and then reconstituted in vitro, both some lyases and some apoproteins were difficult

to dissolve and refold, so reconstitution yields in vitro were often low and poorly

reproducible. An example is the phycoerythrin reconstitution: apo-phycoerythrin is

insoluble when expressed in E. coli, and phycoerythrin biosynthesis is therefore very

difficult to study in vitro. Using the heterologous system in E. coli, both α- and

β-phycoerythrin could be correctly chromophorylated with PEB at C84 sites

(consensus sequence) (Zhao et al., 2007a). (2) In vitro reconstitution is complicated

by the fact that most apo-phycobiliproteins bind phycobilins spontaneously, but with

low fidelity and often with low yield. This results in unwanted products and mixtures

that are difficult to analyze and handle, and complicate enzymatic studies. By using

the heterologous system in E. coli, the spontaneous phycobilin binding is generally

inhibited, and properly chromophorylated subunits of phycoerythrocyanin,

phycocyanin and allophycocyanin were successfully prepared by this way (Zhao et

al., 2006, 2007a, 2007b)

In extending this methology to other system, the expression in prokaryotic or

eukaryotic cells of an appropriate selection of genes encoding enzymes and

apophycobiliprotein subunit-containing fusion proteins could lead to the intracellular

production of constructs carrying specific bilins at unique locations, with broad utility in

addressing a variety of questions in cell biology.

The ability to express in heterologous hosts phycobiliprotein subunits with distinctive

spectroscopic properties, such as holo-PecA (λabsmax = 561 nm and a λF

max = 582 nm),

provides the good potential for use as fluorescence probes in single-molecule

detection and single-cell analysis.


3.2 Structure analysis of reconstituted holo-CpcA

3.2.1 Reconstitation principle of holo-CpcA

PCB is biosynthesized from heme by the action of two enzymes, heme oxygenase

and a BV-reductase. Using endogenous heme as substrate, Tooley et al. (2001)

introduced five genes into E. coli to produce holo-HT-CpcA from Synechocystis

PCC6803 with absorption and fluorescence properties characteristic of the native holo

subunit. Besides the genes for the apoprotein (cpcA) and the lyase (cpcE/F), these

included the two respective genes for PCB synthesis, viz. ho1 and pcyA, from the

same organism (Fig. 3-10).

NH

N HN

COOHCOOH

HN

OOH

A

BC

D

1 2

34

5

6

1819

17

16

15

14

31

7

89

1011

12

13

84Cys

SH

Apo-α -phycocyanin 3Z-phycocyanobilin

Biliverdin αⅨ

NN N

COO-COO-

N O

SCys

H

HA

BC

DO

H

HH

NH

N N

COOHCOOH

HN

OO

H

D

C B

A

Heme HO1

+

CpcE+CpcF

Holo-α -phycocyanin

PcyA

84

Fig. 3-10: Minimal biosynthetic pathway for the production of phycocyanobilin from heme and its addition to the C-phycocyanin apo-α- subunit, CpcA.


3.2.2 Expression and Purification of the holo-CpcA

A similar pathway was established for CpcA from M. laminosus and Anabaena

PCC7120. Expressing pCOLADuet-cpcE-cpcF, pACYCDuet-ho1-pcyA and

pETDuet-cpcA plasmids in strain E. coli BL21(DE3), upon induction with IPTG, the

culture acquired a pronounced blue tint. The His-tagged – holoprotein (holo-HT-CpcA)

was purified by immobilized Ni2+ affinity chromatography, and its spectroscopic

properties were determined after dialysis against 50 mM K-phosphate (pH 7.0). The

absorbance spectrum of holo-HT-CpcA had a λmax = 618 nm and a ratio of QVis/uv =

A618 nm/A367 nm of 5.41 (Fig. 3-11). The corresponding values for Synechococcus

PCC7002 holo-CpcA are 620 nm and 4.22 (Fairchild and Glazer, 1994), for

Synechocystis PCC6803 holo-HT-CpcA are 625 nm and 4.14 (Tooley et al., 2001) or

623 nm and 6.41 (Guan et al., 2007). The absorbance properties of native holo-CpcA

preparations from other cyanobacteria are similar (Sidler, 1994). The differences are

due to the different species, and to a large extent also to the different measurement

conditions. In particular QVis/uv depends on pH and ionic strength (Kupka et al., 2008),

compare e.g. the two values for the holoprotein from Synechocystis PCC6803. For

external comparison, other methods are therefore required. It should be mentioned

that the value of QVis/uv =5.41 is in the upper range and indicates complete

chromophorylation of CpcA. The fluorescence emission maximum of the

holo-HT-CpcA produced in E. coli was at λmax=639 nm (Fig. 3-11), as compared with

642 nm for Synechococcus PCC7002 holo-CpcA (Nakamura et al., 1998), 641 nm

(Tooley et al., 2001) or 648 nm (Guan et al., 2007) for Synechocystis PCC6803

holo-HT-CpcA. The variations are again due to species and experimental differences.

Extinction coefficient ε and fluorescence yield ΦF of reconstituted holo-CpcA have

been determined by the methods given in section 2.2.4.3 and 2.2.4.4. The values for

in vivo reconstituted holo-HT-CpcA (ε618=1.35(±0.01)×105 M-1cm-1 and ΦF = 0.22)

were similar to those of reconstituted holo-HT-CpcA in E. coli from Synechocystis

PCC6803 (εM=127600 M-1cm-1 , ΦF = 0.31) (Tooley et al.,2001), but more different

from those of native holo-CpcA from M. laminosus (ε618=1.08×105 M-1cm-1 , ΦF = 0.65)

(Mimuro et al.,1986). These differences may relate to the different condition used in

the measurements, they may furthermore also be influenced by the His-tag present in

the reconstituted chromoprotein, and by the presence of the residual Ni++. Heavy


atoms are known to complex with bile pigments like PCB (Falk, 1989) and quench

their fluorescence. A more detailed characterization of the reconstitution products was

therefore desirable.

0.00

0.05

0.10

0.15A

bsor

ptio

n [a

.u.]

300 400 500 600 700 8000

100

200

300

Wavelength[nm]

Fluo

resc

ence

[a.u

.]

FIG. 3-11: Spectroscopic properties of Anabaena PCC7120 holo-CpcA expressed and assembled in E. coli. Absorption (solid) (λmax= 618 nm) and fluorescence emission spectra (dashed) (λmax=639 nm) of the chromoprotein purified by affinity chromatography.

Analysis of Coomassie brilliant blue-stained gels after SDS-PAGE of the purified

protein showed only one distinct band of 19.5 kDa (Fig.3-12, lane A) corresponding to

the calculated molecular mass of holo-HT-CpcA (19.5 kDa). Covalent binding of the

chromophore was confirmed upon exposure to Zn2+ and UV illumination, which results

in an orange fluorescence (Fig.3-12, lane a).

FIG. 3-12: SDS-PAGE analysis of Anabaena PCC7120 HT-CpcA expressed and assembled in E. coli, and purified by affinity chromatography. Proteins were visualized by Coomassie brilliant blue staining (left panel) and by the UV excited fluorescence of bilin-bearing polypeptides in the presence of Zn2+ (right panel). Lanes A;a, HT-CpcA expressed and assembled in E. coli strain; lanes B;b, molecular mass standards (from the top, 45, 36, 29, 24, 20 and 14 kDa).

A B a b



The biosynthesized holo-CpcA CD spectrum was similar to that of the native α subunit

of C-phycocyanin of M. laminosus (Mimuro et al., 1986), it shows a strong positive Vis

band at 618 nm (Mimuro:618 nm) and a negative near-UV band at 342 nm (Mimuro:

about 342 nm) (Fig. 3-13). The far-UV CD spectra are typical for α-helical proteins.

This confirms native conformations of both the chromophore and the protein.

300 400 500 600 700 800

20

15

10

5

0

5

10

15

20

CD [a

.u.]

200 220 240 260 280 300

-20

-15

-10

-5

0

5

10

CD [a

.u. ]

Wavelength [nm]

FIG. 3-13: CD spectra of reconstituted holo-CpcA Vis- CD (top) and far-UV CD spectra (bottom) of Anabaena PCC7120 holo-CpcA reconstituted and assembled in E. coli and purified by immobilized Ni2+ affinity chromatography.

3.2.4 HPLC–ESI-MS analysis of chromopeptides from holo-CpcA

RP-HPLC separation of the tryptic digest (Fig. 3-14) on a C18 column gave two main

colored peaks. Peak 1 (tr=16.4 min) had an absorption maximum at 637 nm that is

typical for PCB-containing peptides. Peak 2 (tr= 29.1 min) corresponds to free PCB

according to its retention time and the absorption maximum at 684 nm.


300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

peak 1 peak 2

10 20 30 40

0.0

0.1

0.2

0.3

Abso

rptio

n @

650

nm [a

.u.]


1

2

FIG. 3-14: HPLC and in situ absorption spectra of the tryptic digest ofe reconstituted holo-PecA. C18-column, gradient (80:20 to 60:40) of aqueous formic acid (0.1%, pH 2) and acetonitrile containing 0.1% formic acid.

The molecular weight of the PCB containing peptide (tr=16.4 min) was determined by

ESI mass spectrometry. Sequences of chromophore-containing peptides were

deduced from sequences around the chromophore-binding sites in holo-CpcA from

Anabaena PCC7120. In the native α–subunit of C-phycocyanin, PCB is bound to

cysteine resides 84 of the alpha subunit. There is excellent agreement between the

measured peptide Mr and the values calculated from sequences of alpha subunits of

holo-CPC from Anabaena PCC7120 (Table 3-3, Fig 3-15), which proves correct

binding to the correct site in the E. coli system. The major peaks at 468 and 935m/z

represent doubly and singly charge chromopeptide C(PCB)AR respectively. The

chromopeptide sequence C(PCB)AR confirms chromophore covalent binding to

Cys-84 of the apo-protein. Peak of 587 m/z corresponds to the free chromophore that

is partially lost during MS.


FIG. 3-15: Negative ion mass spectrum by ESI-MS of chromopeptide from reconstituted holo-CpcA. The main peaks 468 m/z and 935m/z represent the doubly and singly charged chromopeptide C(PCB)AR, respectively.

Table 3-3 Major mass peaks of chromopeptide from tryptic digestion of holo-CpcA

Peptide mass Chromopeptide sequence Calc. Expt.

C(PCB)AR [M+2H]2+ 467.86 468.18 C(PCB)AR [M+2H]+ 935.71 935.41

PCB [M+2H]+ 587.29 587.30

3.2.5 NMR analysis of peptic chromopeptides from holo-CpcA

For NMR spectroscopy, again peptic chromopeptides were used. RP-HPLC

separation of pepsin digest (Fig. 3-16) was achieved on a C18 column. Peptides

having retention times at 5.4 and 6.2 min show absorption maxima at 639 nm and

638nm respectively (inset of Fig.3-16) that are typical for PCB-containing peptides.

Samples of the 5.4 min fraction were used for NMR spectroscopy.

Tu CPCA -PCB 16

400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450m/z0

100

%

070730 CPCA 6 (0.206) Cm (3:28) TOF MS ES+ 2.09e5468.705

467.232

428.223

935.412

469.207

469.734

587.303

479.226

487.220

487.724562.383

681.914588.320802.482682.414

936.437

937.451

938.488


300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

peak 1peak 2

4 8 12 16 200.0

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orpt

ion@

650n

m [a

.u.]

Retention time[min]

1

2

FIG. 3-16: HPLC(λdetect=650 nm) and absorption of the chromopeptides obtained from peptic digestion of reconstituted holo-CpcA. HPLC conditions as in Fig. 3-14. Absorption spectra of peak 1 (solid line, λmax=639 nm) and of the peak 2 (dashed line λmax=638 nm)

The 1H-NMR spectrum of this chromopeptide is shown in Fig.3-17. All assignments

refer to the A-ring linkage numbering scheme (see Fig 3-10) and are summarized in

Table 3-4.

7 6 5 4 3 2 1 0

C h e m i c a l s h i f t [ p p m ]

FIG. 3-17: 1-D 1H-NMR spectra of chromopeptide SKC(PCB)AR from reconstituted holo-CpcA in 10 mM TFA/D2O

The NMR-spectrum results from a combination of the chromophore and peptide

signals. In the downfield region of the spectrum, there are three vinylic 10-H, 15-H and

5-H resonances at 7.42, 6.23 and 5.84 ppm, respectively, these peaks are


characteristic for the A-ring-linked α-1 PCB chromophore (Bishop et al., 1986). The

31-H multiplet at 3.48 ppm is downfield shifted due to its proximity to the sulfur atom,

comfirming the attachment at this site. The 31-H is coupled to the 32-CH3 group,

yielding a doublet at 1.27 ppm. Similarly the 2- CH3 signal (1.10 ppm) is coupled to the

2-H proton (2.66 ppm). The 3-H proton (multiplet at 3.06) is coupled to both the 2-H

and 31-H. All these signals are again characteristic of a PCB bound to cysteine at

C-31.

Table 3-4: 1H NMR(600-MHz) assignments for peptide SKC(PCB)AR of reconstituted CpcA-PCB ( 10 mM TFA/D2O)

Chemical shift Multiplicity (JH-H,[Hz])


7.42 s 1 10-H 6.23 s 1 15-H 5.84 s 1 5-H 3.47 m 1 3’-H 3.06 m 1 3-H 2.84 m 4 8,12-CH2CH2COOH 2.66 d (6.50) 1 2-H 2.48 m 4 8,12-CH2CH2COOH 2.08 s 2.00 s 1.98 s

9 7,13,17-CH3

1.93 m 2 18- CH2CH3 1.27 d (7.11) 3 3’- CH3 1.10 d (6.41) 3 2-CH3 0.96 m 3 18- CH2CH3

4.33 m 1 Cys α- CH 2.89 m 2 Cys β- CH2 4.45 dd (6.63, 14.06) 1 Ala α- CH 1.21 d (7.12) 3 Ala β- CH3 4.26 dd (6.46 ,13.65) 1 Arg α- CH 1.47 d (4.65) 2 Arg β- CH2 3.11 m 2 Arg γ- CH2 2.97 m 2 Arg δ- CH2 4.52 t 1 Lys α- CH 1.31 d (7.19) 4 Lys β,δ- CH2 1.35 d (7.11) 2 Lys γ- CH2 4.56 m 1 Ser α- CH 3.79 m 2 Ser β- CH2


The resonances attributed to the aromatic methyls at C-7, -13, and -17, the ethyl

group at C- 18, and the propionic acid methylenes at C-8 and -1 2 are salient features

of the spectra of bile pigments. The three aromatic methyls at C-7, -13, and -17

singlets resonate at 2.08, 2.00 and 1.98 ppm. The ethyl group at C-18 is seen as two

multiplets at 1.93 ppm (18- CH2CH3) and 0.96 ppm (18-CH2CH3). The multiplet

resonances from methylene group of the propionic acid side chains at C-8 and C-12

were found at 2.84 and 2.48 ppm. These results correspond well with the

A-ring-linked α-1 PCB from Synechococcus PCC6801 C-PC (Bishop et al., 1986)

In addition to the chromophore signals, resonances from the following amino acid Ser,

Lys, Cys, Ala, Arg are found in the NMR spectrum. Sequence SKC(PCB)AR was

deduced from sequences around the chromophore-binding sites in holo-CpcA from

Anabaena sp. strain PCC7120.

3.2.6 Discussion

The studies described here documented successful reconstitution of the predicted

pathway for the biosynthesis of the phycocyanin holo-α subunit (Fig.3-10) in E. coli.

The results of the experiments support and extend the conclusion reached by Tooley

et al. (2001, in vivo), Guan et al. (2007, in vivo), Zhao et al. (2006, in vitro) and

Fairchild et al. (1992, in vitro) that the heterodimeric lyase/isomerase (CpcE/CpcF)

catalyzes the covalent attachment of PCB. They unambiguously identify the correct

binding site. The lack of split NMR signals, which is particularly clear in the low-field

region, establishes that no isomers are formed in the process, and the coincidence

with chromophore signals of a PCB peptide from holo-CpcA of Synechocystis

PCC6803 also verify the correct stereochemistry of the product.

SDS-PAGE gel (Fig. 3-12) showed that there is a single band (holo-HT-CpcA) at 19.5

kDa, while Tooley’s work yielded two separate bands for holo and apo-HT-CpcA

(Tooley et al., 2001), probably due to incomplete chromophore attachment. Complete

chromophorylation in the current reconstitution is also supported by the QA value.

The evidence that CpcE and CpcF are needed for the correct addition of PCB to

apo-CpcA, in vivo in various cyanobacteria (Zhou et al., 1992; Swanson et al.,

1992;Cai et al., 1997; Tooley et al., 2001) as well as in vitro (Fairchild et al., 1992,


Zhao et al., 2006), is very strong. In vitro, nonenzymic addition of PCB to CpcA leads

to the formation of product mixtures containing an unnatural, oxidized adduct,

mesobiliverdin-CpcA, with distinctive spectroscopic properties (Arciero et al., 1988);

however, normal holo-CpcA is produced in the presence of CpcE and CpcF (Fairchild

et al., 1992). In vivo nonenzymic addition of PCB to CpcA leads to the formation of

very little of the natural product (0.2%) (Chen, 2006).

In vitro, CpcE and CpcF overproduced in E. coli catalyze the attachment of PCB to

the α subunit of apophycocyanin at the appropriate site, α-Cys-84, to form the

correct adduct. CpcE and CpcF also efficiently catalyze the reverse reaction, in

which the bilin from holo-α subunit is transferred either to the apo-α subunit of the

same CPC or to the apo-α subunit of a heterologous CPC. The forward and reverse

reactions each require both CpcE and CpcF and are specific for the α-Cys-84

position (Fairchild et al., 1992).

CpcE and CpcF form an enzymatically active 1:1 complex (CpcEF), stable to size

exclusion chromatography. CpcEF causes a reduction in α-PC fluorescence and

strongly affects its absorption spectrum but has no effect on the β subunit. CpcEF also

catalyzes the addition of PEB to apo-alpha PC; PEB is thought to be on the

biosynthetic pathway of PCB. CpcEF shows a preference for PCB relative to PEB,

both in binding affinity and in the rate of catalysis, sufficient to account for selective

attachment of PCB to apo-α C-PC (Fairchild and Glazer 1994).

The CpcE ⁄F bound PCB could be transferred to CpcA to yield holo-CpcA. The PCB

transfer capacity correlates with the activity of the lyase, indicating that PCB bound to

CpcE/F is an intermediate of the enzymatic reaction. A catalytic mechanism has been

proposed, in which a CpcE/F complex binds PCB and adjusts via a salt bridge the

conformation of PCB, which is then transferred to CpcA (Zhao et al., 2006b). In

summary, these results show that in E.coli the presence of CpcE and CpcF is

necessary and sufficient to correctly chromophorylate CpcA with PCB, and that

autocatalytic or spontaneous chromophylation is negligible. The latter is in contrast to

reconstitution in vitro.


3.3 Structure analysis of reconstituted subunits of

allophycocyanin

3.3.1 Reconstitution principle of subunits of allophycocyanin


heme oxygenase and BV-reductase. PCB is then attached to the apoprotein of

allophycocyanin by a lyase, CpcS (alr0617) (Zhao et al. 2007). Using endogenous

heme as substrate, Zhao et al.(2007) introduced four genes into E. coli to produce

holo-subunits of allophycocyanins from Anabaena PCC7120 with spectroscopic

properties characteristic of the native holo subunits. Besides the genes for the

apoproteins (apcA1, apcB, apcA2, apcD, apcF) and the lyase (cpcS), these included

the two respective genes for PCB synthesis, viz. ho1 and pcyA, from the same

organism(Fig.3-18)

NH

N HN

COOHCOOH

HN

OOH

A

BC

D

1 2

34

5

6

1819

17

16

15

14

31

7

89

1011

12

13

84Cys

SH

Holo-α /β -allophycocyanin

NN N

COO-COO-

N O

SCys

H

H

H

OH

H

Apo-α /β -allophycocyanin 3Z-phycocyanobilin

Biliverdin αⅨ

NH

N N

COOHCOOH

HN

OO

H

D

C B

A

Heme HO1

+

CpcSPcyA

84

Fig. 3-18: Minimal biosynthetic pathway for the production of PCB from heme, and addition to the allophycocyanin apo- subunits.


3.3.2 Expression and Purification of the subunits of allophycocyanin

Plasmids pCDFDuet-cpcS, pACYCDuet-ho1-pcyA and pET-apcA1 (or apcB, apcA2,

apcD, apcF) were expressed in the E. coli strain BL21(DE3). Upon induction with

IPTG, the culture acquired a pronounced blue tint. The various His-tagged subunits of

APC were purified by affinity chromatography. The absorption and fluorescence

emission properties of the dialyzed chromoproteins are shown in. Fig. 3-19 and Tab.

3-5.

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

50

100

150

200

250

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

50

100

150

200

250

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

100

200

300

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.04

0.08

0.12

Abs

orpt

ion

[a.u

]

Wavelength [nm]

0

50

100

150

200

250

300Fl

uore

scen

ce [a

.u.]

0

50

100

150

200

Fluo

resc

ence

[a.u

]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

A B

C D

E

FIG. 3-19: Absorption(solid) and fluorescence emission(dashed) properties of Anabaena PCC7120 PCB-ApcA1(A), PCB -ApcB(B), PCB -ApcA2(C), PCB -ApcD(D) and PCB -ApcF(E) expressed in E. coli. Table 3-5: Quantitative absorption and fluorescence data of the biosynthesized and purified PCB-ApcA1, PCB-ApcB, PCB-ApcA2, PCB-ApcD, and PCB-ApcF.

Absorption Fluorescence Biliprotein λmax [nm] (QVis/uv) εVis [M-1•cm]-1 λmax [nm] ΦF

PCB-ApcA1 338/618 (4.3) 111,000 641 0.37 PCB-ApcB 344/613 (2.4) 86,700 640 0.20

PCB-ApcA2 365/622 (3.2) 71,800 641 0.18 PCB-ApcD 346/650 (2.3) 62000 663 0.074 PCB-ApcF 355/622 (2.4) 96,400 645 0.19


The absorption and fluorescence emission data for PCB-ApcA1 and PCB-ApcB agree,

qualitatively and quantitatively, with those of the isolated APC subunits, α-APC and

β-APC, respectively, from several cyanobacteria (MacColl, 2004; Rümbeli et al., 1987;

Canaani et al., 1980). The corresponding values for Synechocystis PCC6803 ApcA

reconstituted by CpcE/CpcF catalysis are 626 nm and 640 nm (Yang et al., 2008).

The differences are in part due to the different species and lyases, and to a large

extent also to the different measurement conditions. All products showed the correct

chromopeptide sequences for APC isolated from Anabaena PCC7120 (Fig. 3-21).

The absorption and fluorescence of autocatalytic reconstituted ApcA from Spirulina sp.

are 610 nm and 642 nm respectiverly (Hu et al., 2006), which is rather different

spectroscopically, maybe is not properly attached.

The heterologous reconstitution product PCB-ApcA2 had similar absorption and

fluorescence spectra as PCB-ApcA1. The strong fluorescence (ΦF=0.18) and the

large intensity ratio of the Vis- and UV-absorptions (QA=3.2) are characteristic of a

native biliprotein. ApcA2 is highly homologous to ApcA1, but to our knowledge it has

never been isolated, and may be an allophycocyanin-like protein (Montgomery et al.,

2004).

The absorption (622 nm) and fluorescence maxima (645 nm) of PCB-ApcF are very

similar to the values reported for the native 16.2 kDa β-subunit of Mastigocladus

PCC7603 (Rümbeli et al., 1987).

The strong absorption (650 nm) and the relatively weak fluorescence (663 nm) of

PCB-ApcD agree well with those of the native α-subunit of APB from Synechococcus

6301 (Lundell and Glazer, 1981, λabs=648 nm). This maximum is significantly blue

shifted from that found for the intact trimer containing the αB subunit, which has a

broad band from 671 to 679 nm. The spectral differences between APC trimers and

isolated subunits are well known, but their origins are unclear. There are several

possible reasons, excitonic coupling, modified interactions with the protein, and linker

proteins. The αB subunit attains its complete trimeric spectrum by engaging in strong

coupling with the chromophore on the allophycocyanin β subunit. The full red shift for

the trimer containing the αB subunit would consist of two parts; one part provided by


the αB subunit itself, and the second part coming from another strongly coupled

chromophore. In support of this possibility, the amino acid sequence of the αB subunit

shows a significant homology with the α subunit of allophycocyanin(ApcA), and it

might readily replace the allophycocyanin a subunit in a similar type of strong coupling

(MacColl, 2004).

It is concluded that in the E. coli system a single lyase, CpcS, attaches PCB to

Cysteine-84 (consensus sequence) of ApcA1, ApcB, ApcA2, ApcD and ApcF to form

holo-APC units with optical spectra like those of the respective native chromoproteins.


CD spectra are very sensitive to the conformation of protein and chromophore: they

were therefore measured for reconstituted and FPLC-purified PCB-ApcA1, PCB-ApcB,

PCB-ApcA2, PCB-ApcD and PCB-ApcF (Fig. 3-20 and in Tab. 3-6).

The CD spectrum of PCB-ApcA1 (614(+)/340(-) nm), PCB-ApcB (614(+)/341(-) nm),

PCB-ApcA2 (384(+)/630(-) nm), PCB-ApcD (588(+)/666(-) nm) and PCB-ApcF

(617(+)/343(-) nm) are typical for PCB-containing biliproteins. However, the CD

signals of ApcA2 and ApcD are of opposite sign, indicating a different chromophore

conformation. Spectrum of ApcD also shows a broad positive band, indicating a

heterogeneity. The far-UV CD spectra of PCB-ApcA1, PCB-ApcB, PCB-ApcD and

PCB-ApcF are typical for α-helical proteins. This confirms native conformations of

both the chromophore and the protein for ApcA1, ApcB, and ApcF. The far-UV CD

spectrum of PCB-ApcA2 is typical for a β-strand protein, no such case has been

reported so far for biliproteins, it may therefore be due to misfolding; this protein has

never been isolated from Anabaena PCC 7120.


Table 3-6: CD bands of biliproteins

300 400 500 600 700 800-8

-4

0

4

8

CD [a

.u.]

200 220 240 260 280 300

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

CD [a

.u.]

Wavelength [nm]

300 400 500 600 700 800-4

-3

-2

-1

0

1

2

3

CD [a

.u.]

200 220 240 260 280 300-5.0-4.5-4.0-3.5-3.0-2.5-2.0-1.5-1.0-0.50.00.5

CD [a

.u.]

Wavelength [nm]

300 400 500 600 700 800

-10

-5

0

5

10

15

CD [a

.u.]

200 220 240 260 280 300-2.0

-1.5

-1.0

-0.5

0.0

CD [ a.u.]

Wavelength [nm]

300 400 500 600 700 800

-12

-8

-4

0

4

8

12

16

CD [a

.u.]

200 220 240 260 280 300-3

-2

-1

0

CD [ a.u.]

Wavelength [nm]

200 220 240 260 280 300

-5

-4

-3

-2

-1

0

CD [ a.u.]

Wavelength [nm]

300 400 500 600 700 800

-15

-10

-5

0

5

10

15

20

CD [ a.u.]

A B

C D

E

FIG. 3-20: CD spectroa of reconstituted and FPLC-purified PCB-ApcA1(A), PCB-ApcB(B), PCB-ApcA2(C), PCB-ApcD(D) and PCB-ApcF(E) CD spectra, Vis- CD (top) and far-UV CD spectra (bottom) of Anabaena PCC7120 subunits of allophycocyanin reconstituted with PCB in E. coli and FPLC-purified, in KPB(20 mM, pH 7.0) containing NaCl (500 mM)

3.3.4 Chromophore analyses of reconstitution products

After denaturation in acidic urea solution (8 M, pH 2.0), the purified reconstituted

chromoproteins (PCB-ApcA1, -ApcB, -ApcA2, -ApcD and -ApcF) gave nearly identical

spectra with maximal absorption at 661 - 664 nm. The spectrum of denatured ApcB is

Biliprotein Visible CD (λmax[nm]) UV CD (λmin[nm])

PCB-ApcA1 614 (+) 340 (-) 210/221 PCB-ApcB 614 (+) 341 (-) 211/221

PCB-ApcA2 630 (-) 384 (+) 212 PCB-ApcD 666 (-) 588 (+) 210/222 PCB-ApcF 617 (+) 343 (-) 210/222


shown as an example in Fig.3-21. This is evidence for an intact, cysteine-bound PCB

and the absence of significant amounts of mesobiliverdin, which absorbs at longer

wavelengths (Arciero et al., 1988a, b). Purified PCB-ApcA1 and PCB-ApcB were

digested with pepsin under acidic conditions, chromopeptides enriched by

chromatography on Bio-Gel under acidic conditions, and then analyzed by HPLC

(Storf, 2003). The chromopeptides absorb maximally at 656 nm (in dilute HCl)

(Fig.3-21), which is characteristic for PCB-chromopeptides (Arciero et al., 1988b;

Zhao et al., 2006). HPLC analyses gave in both cases one major and several minor

peaks (Fig. 3-21), which were at identical positions to the respective ones arising from

APC isolated from the parent cyanobacterium, Anabaena PCC7120. In summary,

therefore, we conclude that CpcS catalyzes correct attachment of PCB to both ApcA1

and ApcB to form holo-α-APC and β-APC, respectively.

5 10 15 200.000

0.007

0.014

0.000

0.012

0.024

0.000

0.012

0.024

Abs

orpt

ion

Retention Time [min]

Native APC

PCB-ApcAB

PCB-ApcB

300 400 500 600 700 800

0.02

0.04

0.06

Abs

orption

Wavelength [nm]

A

FIG. 3-21: A: Absorption (solid line, λmax = 662 nm) of the purified chromoprotein, ApcB in acidic urea (8 M, pH 2.0), and of the purified peptide obtained by pepsin digestion, in diluted HCl (pH 2.5, dashed line, λmax = 656 nm); PCB-ApcA1 produced similar results. B: HPLC (λdetect = 650 nm) of the chromopeptides obtained from peptic digestion of the reconstituted PCB-ApcA1 (top), of the reconstituted PCB-ApcB (middle), and of native APC from Anabaena PCC7120 (bottom). Reconstitutions were heterologously done in E. coli with genes from Anabaena PCC7120.


3.3.5 HPLC–ESI-MS analysis of chromopeptides from reconstituted

subunits of allophycocyanin

RP-HPLC separation of trypsin digests (Fig.3-22) on a C18 column, gave two types of

colored components. Component 1 (A:tr=24.6 min and tr=27.7 min ; B: tr=26.7 min and

tr=27.1 min; C: tr=27.7 min ; D: tr=28.2 min; E: tr=20.6 min and tr=23.8 min) had an

absorption maximum around 640 nm (Fig.3-23) that is typical for PCB-containing

peptides; component 2 (A:tr=39.3 min ; B: tr=40.7 min; C: tr=39.1 min ; D: tr=39.2 min;)

corresponds to free PCB according to its retention time and the absorption

maximum about at 685 nm (Fig.3- 23). The underlined fractions were used for MS.

10 20 30 40 500.00

0.05

0.10

0.15

0.20

0.25

0.30

Abs

orpt

ion

[a.u

.]


1

2

10 20 30 40 500.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Abs

orpt

ion

[a.u

.]

Retention time [min]10 20 30 40 50

0.00

0.01

0.02

0.03

0.04

Abs

orpt

ion

[a.u

.]


1 2

10 20 30 40 500.00

0.05

0.10

0.15

0.20

Abs

orpt

ion

[a.u

.]


1

2

3

10 20 30 40 500.00

0.02

0.04

0.06

Abs

orpt

ion

[a.u

.]


1

2 3A

C

E

B

D

FIG. 3-22: HPLC of chromopeptides obtained from tryptic digestion of the reconstituted holo-subunit of allophycocyanin(PCB-ApcA1(A), PCB-ApcB(B), PCB-ApcA2(C), PCB-ApcD(D) and PCB-ApcF(E)). HPLC were eluted using a gradient (80:20 to 60:40) of formic acid (0.1%, pH 2) and acetonitrile containing 0.1% formic acid.


300 400 500 600 700 8000.0

0.1

0.2

0.3

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

peak 1 peak 2

300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

Abs

orpt

ion

[a.u

.]

Wavelength [nm]300 400 500 600 700 800

0.00

0.01

0.02

0.03

0.04

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

peak 1 peak 2

300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

0.25

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

peak 1 peak 2 peak 3

300 400 500 600 700 8000.00

0.01

0.02

0.03

0.04

0.05

0.06

Abs

orpt

ion

[a.u

.]

Wavelength [nm]


A B

C D

E

FIG. 3-23: Absorption spectra of the chromopeptides obtained from tryptic digestion of the reconstituted holo-subunit of allophycocyanin (PCB-ApcA1(A), PCB-ApcB(B), PCB-ApcA2(C), PCB-ApcD(D) and PCB-ApcF(E))..

Sequences of potential chromophore-containing peptides were deduced from

sequences around the chromophore-binding sites in subunits of allophycocyanin from

Anabaena PCC7120. In native APC subunits, PCB is bound to cysteine resides 81 of

the α-subunit and 82 of the β-subunit. There is excellent agreement between the

measured peptide Mr values and peptide Mr values calculated from sequences of

alpha subunits and beta subunits of allophycocyanin from Anabaena PCC7120 (Table

3-7). The chromopeptide sequences confirm the attachment of PCB to the known

binding sites (C84, consensus sequence) of ApcA1 or ApcB, ApcD and ApcF also to

the consensus site of Apc A2.


Table 3-7: Mass of chromopeptides from tryptic digestion Peptide peak (m/z) Biliprotein Chromopeptide Calc. Expt.

PCB-ApcA1 R(62)PDVVSPGGNAYG QEMTATC(PCB)LR

[M+4H]4+ 727.47 727.86

PCB-ApcA2 R(62)PDIVSPGGNAYGQDMTATC(PCB)LR

[M+3H]3+ 969.96 970.09

PCB-ApcB Y(79)AAC(PCB)IR [M+2H]2+ 641.56 641.78 PCB-ApcD A(79)LC(PCB)IR [M+2H]2+ 581.02 581.28 PCB-ApcF L(79)AAC(PCB)LR [M+2H]2+ 616.56 616.78

3.3.6 NMR analysis of peptic chromopeptides from reconstituted

subunits of allophycocyanin

RP-HPLC separation of pepsin digests (Fig.3-24) on a C18 column, gave one type of

colored components, including A:tr=25.5 min and tr=26.6 min ; B: tr=17.9, 18.7, 19.6

and 23.9 min; C: tr=25.2 min ; D: tr=18.7, 19.8 and 23.1 min; E: tr=19.9, 26.9 and 27.9

min, had an absorption maximum about at 640 nm (Fig.3-25) that is typical for

PCB-containing peptides. The underlined fractions were used to measure NMR

spectra.

10 20 30 40 500.0

0.2

0.4

0.6

0.8

Abs

orpt

ion [a.u.]

Retention time(min)

1

2

10 20 30 40 500.0

0.2

0.4

0.6

Abs

orpt

ion [a.u]


1

2

3

10 20 30 40 500.0

0.1

0.2

0.3

Abs

orpt

ion [a.u.]


1

2

3

10 20 30 40 500.0

0.2

0.4

0.6

Abs

orpt

ion [a.u.]


1

2

3

4

10 20 30 40 500.0

0.2

0.4

0.6

0.8

Abs

orpt

ion [a.u.]


1

2A B

C D

E

FIG. 3-24: HPLC of chromopeptides obtained from peptic digestion of the reconstituted holo-subunit of allophycocyanin(PCB-ApcA1(A), PCB-ApcB(B), PCB-ApcA2(C), PCB-ApcD(D) and PCB-ApcF(E))..


300 400 500 600 700 8000.0

0.2

0.4

0.6

Abs

orption [a.u.]

Wavelength [nm]


300 400 500 600 700 8000.0

0.1

0.2

0.3

Abs

orption [a.u.]

Wavelength [nm]


300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7Abs

orption [a.u.]

Wavelength [nm]

peak 1 peak 2

300 400 500 600 700 8000.0

0.2

0.4

0.6

Abs

orption [a.u.]

Wavelength [nm]

peak 1 peak 2 peak 3 peak 4

300 400 500 600 700 8000.0

0.2

0.4

0.6

Abs

orption [a.u]

Wavelength [nm]

peak 1 peak 2A B

C D

E

FIG. 3-25: Absorption spectra of the chromopeptides obtained from peptic digestion of the reconstituted holo-subunit of allophycocyanin(PCB-ApcA1(A), PCB-ApcB(B), PCB-ApcA2(C), PCB-ApcD(D) and PCB-ApcF(E)).

In order to determine the chromophore structure and mode of linkage to the peptide,

the 600-MHz 'H NMR spectra of the chromopeptide of holo subunit of allophycocyanin

(PCB-ApcA1, PCB-ApcB, PCB-ApcA2 and PCB-ApcF) in 10 mM TFA/D2O, were

compared (Table 3-8,9,10 and Fig. 3-26) .

8 7 6 5 4 3 2 1 0


8 7 6 5 4 3 2 1 0

8 7 6 5 4 3 2 1 0

8 7 6 5 4 3 2 1 0

A

B

C

E

FIG. 3-26: 1-D 1H NMR spectra of chromopeptides from holo subunit of allophycocyanin(PCB-ApcA1(A), PCB-ApcB(B), PCB-ApcA2(C) and PCB-ApcF(E)) in 10 mM TFA/D2O at 25 °C


Table 3-8: 1H NMR(600-MHz) assignments for chromohore protons in APC chromopeptides of reconstituted ApcA1-PCB (A), ApcB-PCB(B), ApcA2-PCB(C), ApcF-PCB(E)( 10 mM TFA/D2O).

Parallel analysis of the NMR spectra of holo subunit of allophycocyanin (PCB-ApcA1,

PCB-ApcB, PCB-ApcA2 and PCB-ApcF) chromopeptide reveals the expected

similarity of the resonant frequencies of the chromophore (Table 3-8), the assignment

of almost all resonances to the phycocyanobilin (PCB) prosthetic group can be made.

The 1H NMR spectral assignments of the bilin moiety of chromopeptides show great

similarity with those reported for the blue pigment (=PCB-peptide) (Bishop et al.,

1986). In particular, the resonances attributed to the aromatic methyls of C-7, -13, and

-17, the three vinylic of C-5, -10, and -15, the ethyl group of C-18, and the propionic

acid methylenes at C-8 and -12 are salient features of the spectra of both pigments.

Parallel analysis of the NMR spectra of PCB-ApcA1 and PCB-ApcA2 chromopeptide

reveals the expected similarity of the resonant frequencies of the amino acid residues

epected for binding at Cysteines-81 (Table 3-9). These two α-apoproteins are highly

homologous (86% similar, 70% identical). All amino acid α protons appear between

3.7 and 4.7 ppm. The chemical shifts of the hydrogens of these same amino acids

(Ala, Cys, Gly, Gln, Met and Thr) in the PCB-ApcA1 chromopeptide are displaced

within 0.04 ppm upfield from those in PCB-ApcA2 chromopeptide. However, these

Chemical shift (ppm) A B C E

Multiplicity(JH-H,[Hz])

Number of H’s

Assignment

6.92 6.98 7.01 7.03 s 1 10-H 6.22 6.23 6.23 6.23 s 1 15-H 5.78 5.74 5.79 5.80 s 1 5-H 3.47 3.46 3.46 3.46 m 1 3’-H 2.92 3.02 2.92 2.99 m 1 3-H 2.85 2.85 2.86 2.88 m 4 8,12-CH2CH2COOH 2.47 2.46 2.51 2.48 m 1 2-H 2.32 2.35 2.34 2.34 m 4 8,12-CH2CH2COOH2.07 2.06 2.07 2.06 s 1.98 1.98 1.98 1.98 s 1.83 1.84 1.83 1.83 s

9 7,13,17-CH3

1.96 1.96 1.96 1.95 m 2 18- CH2CH3 1.35 1.38 1.34 1.34 d (6.73) 3 3’- CH3 1.15 1.17 1.15 1.12 d (6.94) 3 2-CH3 0.95 1.11 0.95 0.94 t (7.54) 3 18- CH2CH3


chemical shifts are not observed in some of the resonances of the γ protons of Gln,

Glu and Met of chromopeptides. Minor differences in the spectra of the two

chromopeptides are evident in the resonances attributed to the Glu and Asp residues.

Based on the sequences around the chromophore-binding sites in PCB-ApcA1 and

PCB-ApcA2 from Anabaena PCC7120, chromopeptides sequence GQEMTATC(PCB )

of PCB- ApcA1 and GQDMTATC(PCB) of PCB-ApcA2 were deduced.

Table 3-9: 1H NMR(600-MHz) assignments for peptides of reconstituted ApcA1-PCB and ApcA2-PCB ( 10 mM TFA/D2O).

Parallel analysis of the NMR spectra of PCB-ApcB and PCB-ApcF chromopeptides

reveals the expected similarity of the resonant frequencies of the corresponding

amino acid residues (Table 3-10). They are β subunits of allophycocyanin, but their

homology is relatively low (72% similar, 51% identical). All amino acid α protons

appear between 3.8 and 4.6 ppm. The chemical shifts of the α hydrogens of like

amino acids (Ala, Cys) in PCB-ApcB chromopeptide are displaced within 0.04 ppm

upfield from the respective ones in the PCB-ApcF chromopeptides. Minor differences

(0.1-0.2 ppm) in the spectra of the two chromopeptides are evident in the resonances

Chemical shift ApcA1-PCB ApcA2-PCB


Number of H’s

Assignment

3.75 3.75 s 2 Gly α- CH2

4.17 4.19 d (7.28) 1 Gln α- CH 2.28 2.27 m 2 Gln β- CH2

/ / / 2 Gln γ- CH2 3.94 3.95 t (6.55) 1 Met α- CH 2.17 2.14 m 2 Met β- CH2

/ / / 2 Met γ- CH2 4.42 4.43 m 1 Cys α- CH 2.99 3.03 m 2 Cys β- CH2

4.08,3.85 4.12,3.87 m 2 Thr α- CH 3.52 3.56 d (6.93) 2 Thr β- CH 1.24 1.23 d (6.94) 6 Thr γ- CH3 4.01 4.01 q (6.95) 1 Ala α- CH 1.24 1.23 d (6.93) 3 Ala β- CH3 4.12 / d (4.19) 1 Glu α- CH 2.33 / m 2 Glu β- CH2

/ / / 2 Glu γ- CH2 / 4.61 m 1 Asp α- CH / 2.86 m 2 Asp β- CH2


attributed to the Thr and Arg residues, and chemical shifts are not observed for some

of the resonances of the β and δ protons of Arg. Major differences in the spectra of the

two chromopeptides are evident in the resonances attributed to the Asp, Ile, Leu and

Tyr residues. Based on the sequences around the chromophore-binding sites in

PCB-ApcB and PCB-ApcF from Anabaena PCC7120, chromopeptides sequence

TTRRYAAC(PCB)IRD of PCB- ApcB and TTRRLAAC(PCB) of PCB-ApcF were

deduced.

Table 3-10: 1H NMR(600-MHz) assignments for peptides of reconstituted ApcB-PCB and ApcF-PCB (10 mM TFA/D2O).

3.3.7 Discussion

The studies described here document the successful reconstitution of the predicted

Chemical shift ApcB-PCB ApcF-PCB


Number of H’s

Assignment

4.18, 4.27 3.83, 4.08 m 1 Thr α- CH 3.47 3.54 m 1 Thr β- CH

1.24, 1.31 1.24, 1.29 d (6.92) 3 Thr γ- CH3 4.02, 4.22, 4.27 4.08 m 1 Arg α- CH

/ / / 2 Arg β- CH2 1.31 1.35 d (6.95) 2 Arg γ- CH2

/ / / 2 Arg δ- CH2 4.39 4.38 t (7.05) 1 Cys α- CH 3.11 3.06 m 2 Cys β- CH2 4.56 / d (8.23) 1 Asp α- CH 2.85 / m 2 Asp β- CH2

/ 4.08 m 1 Leu α- CH / 1.45 m 2 Leu β- CH2 / 0.78 d (5.91) 1 Leu γ- CH / 0.75 d (6.23) 3 Leu δ- CH3

3.84, 4.02 3.83, 4.01 q (7.04) 1 Ala α- CH 1.24, 1.31 1.24,1.29 d (6.92) 3 Ala β- CH3

4.48 / t (6.72) 1 Tyr α- CH 2.85 / m 2 Tyr β- CH2 4.35 / d (4.96) 1 Ile α- CH 0.86 / m 1 Ile β- CH 1.31 / d (6.95) 2 Ile γ- CH2 0.78 / m 3 Ile δ- CH3


pathway for the biosynthesis of the subunits of allophycocyanin (Fig.3-18) in E. coli,

and identify CpcS not only as a lyase for Cys-84 of the β subunits of phycocyanin and

phycoerythrocyanin in Anabaena PCC 7120 (Zhao et al., 2006a), but also for all APC

subunits. They unambiguously identify the correct binding site. The lack of split

signals in the NMR spectra, which is particularly clear in the low-field region,

establishes that no isomers are formed in the process, and the coincidence with

chromophore signals of PCB peptides from subunits of allophycocyanin of Anabaena

PCC7120 also verify the correct stereochemistry of the product. Taken together,

these results demonstrate, in the E. coli system, a remarkable substrate spectrum of

CpcS: besides the already known β-subunits of CPC and PEC (Zhao et al., 2006; 3.4

and 3.5), it covers all APC-subunits known, and an additional α-subunit that has not

been described before. The enzyme has, on the other hand, a high site-specificity: in

CpcB and PecB, PCB is attached exclusively to the Cys-84 binding site, which is

homologous to the Cys-84 (consensus sequence) binding site of the APC subunits.

Allophycocyanin is a biliprotein located in the core of the phycobilisome. The

biliprotein is isolated and purified as a trimer (α3β3), where a monomer has αβ

structure. Each α and β subunit have a single PCB chromophore. The trimer of

allophycocyanin has an unusual absorption maximum at 650 nm with a shoulder at

620 nm, while the monomer has an absorption maximum at 615 nm. There are three

other core proteins with PCB chromophores, namely, LCM (ApcF), β16 (ApcF), and αB

(ApcD) (MacColl, 2004). Ashby and Mullineaux (1999) suggest that both ApcD and

ApcF are involved in the energy transfer from phycobilisome to PS II and PS I. In both

cases, the energy transfer to the reaction centers may be eventually via the

chromophore of ApcE (the Lcm or anchor polypeptide). However, the major route of

energy transfer to both kinds of reaction centre appears to involve ApcF rather than

ApcD. When both ApcF and ApcD are absent, the phycobilisomes are unable to

transfer energy to either reaction centre.

The report of Shen et al. (2006) on the CpcSTUV-family opened a new horizon in

biliprotein biosynthesis. It indicated that these were lyases with a much broader

specificity than that of the known E/F-type, extending to APC, and that the substrate

specificity was controlled by the relative amounts of the different lyases present. Their

suggestions have been substantiated, but also modified for one member, CpcS, by

the present study. The substrate specificity is even broader; it can not only attach


PCB to all APCs but also PEB to both subunits of CPE (Zhao et al., 2007). Together

with its already established action on the ß-subunits of CPC and PEC (Zhao et al.,

2006), CpcS is therefore capable of attaching chromophores to members of all groups

of biliproteins. Particularly unexpected was the chromophorylation of CpeA at

cysteine-84, because an E/F-type lyase had been suggested to work on this protein

(Kahn et al., 1997). At the same time, the specificity is high with regard to the binding

site, which is always cysteine-84(consensus sequence). In APC proteins, this is the

only binding site, but the selectivity holds also for the proteins with multiple binding

sites. A similar specificity had already been shown for the CPC and PEC subunits,

CpcB and PecB, which both carry two binding sites (Zhao et al., 2006a). CpcS

therefore seems to be a (nearly) universal lyase with respect to the protein, but is at

the same time highly specific for a single binding site, cysteine-84. Similar results

were more recently reported by Shen et al. (2008) and Saunee et al. (2008) for CpcS-I

and CpcU. However, in this case CpcS-I and CpcU form a heterodimeric lyase that

attaches PCB to Cys-82 on β-PC and to Cys-81 of the α and β subunits of AP from

Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002.

Homologs of the CpcS and CpeS proteins can be classified into five groups. Two of

them, groups A and B, only include strains that can synthesize one or more PEs. This

observation, and the further observation that these genes are usually clustered with

other genes known to play a role in PE biosynthesis or assembly, suggests that these

proteins denoted CpeS and CpeU, respectively, probably play roles in

phycoerythrobilin (PEB) attachment to PE subunits. All of the other groups contain at

least some members that only synthesize proteins with PCB chromophores, and thus

these proteins cannot be involved in PE synthesis, at least in some organisms. The

largest of these groups, Group C, can be further divided into three clades, which

Saunée et al.(2008) have denoted CpcS-I, CpcS-II, and CpcS-III. Synechococcus sp.

PCC 7002 has a protein within the CpcS-I clade. CpcS-I and CpcU together form a

heterodimeric PCB lyase for β-PC and AP subunits. Clade III of Group C includes the

Anabaena sp. PCC 7120 open reading frame alr0617, the product of which has been

shown by this work (Zhao et al.,2006a, 2007a) to ligate PCB to Cys-82 of CpcB, PecB,

ApcA, ApcB, ApcD, and ApcF. With the exception of N. punctiforme, no organism with

a CpcS-III protein also has a protein in Group D (CpcU). The CpcS-II clade includes a

variety of marine Synechococcus sp., some of which produce PC with PEB


chromophores. Finally, Group E includes several divergent sequences, and the

organisms that produce the members of this clade do not have an obvious PBP

pigmentation pattern. Synechococcus sp. PCC 7002 produces one of the members of

this clade; we have provisionally designated these proteins as CpcV (Shen et

al.,2008).

There is one notable exception for the protein specificity. CpcA and PecA both have a

cysteine-84 binding site, but here CpcS is inactive (Zhao et al., 2006), and the sites

are rather served by the site and protein-specific EF-type lyases (Schluchter et al.,

1999; Zhao et al., 2000). This exception, and the presence of specialized enzymes for

only these subunits, indicates some special status of the latter. One might argue that

in the case of PecA, the EF-type lyase has a second function by which the

chromophore is isomerized (Arciero et al., 1988). However, such a function is not

required for CpcA. Here, is attached by homologous E/F-type lyases in the

conventional fashion by addition to the Δ3,31-double bond, and with stereochemistries

and conformations that are very similar to those of PCB bound to cysteine-84 of the

CPC and PEC ß-subunits (Fairchild et al., 1994; Zhao et al., 2005; Dammeyer et al.,

2006). This indicates a structural difference which, among the cysteine-84 binding

sites, sets aside the ones of CpcA and PecA from those of the other biliproteins. Such

a difference is supported by a sequence analysis (Zhao et al., 2007a).

It is tempting to speculate on a functional difference of these two groups of biliproteins

that goes beyond the lyase specificity. The α-84 sites of all cyanobacterial and red

algal biliproteins are at the contact surface to the β-subunits in the trimers, and show

similar geometries and stereochemistries. While this argues against a structural or

photosynthetic function, the difference may be relevant to phycobilisome degradation.

The α-subunit of EF-type lyases share homologies to proteins involved in

phycobilisomes degradation (Dolganov et al., 1999; Balabas et al., 2003). They are

also capable of chromophore detachment (Schirmer et al., 1987), while no such

activity has been found for CpcS (Zhao et al., 2006a). There is, moreover, another

protein interaction that is specific for these subunits: complex formation of α-CPC and

α-PEC has been observed with NblA, which is implicated in biliprotein degradation

(Luque et al., 2003; Bienert et al., 2006).


3.4 Structure analysis of reconstitution β-subunit of

C-phycocyanin

3.4.1 Reconstitution principle of β-subunit of C-phycocyanin


heme oxygenase and BV-reductase. PCB is then attached to the β subunit apoprotein

of C-phycocyanin by a lyase, CpcS (Zhao et al. 2006; Shen et al., 2008) or CpcT

(Zhao et al. 2007; Shen et al. 2006). Using endogenous heme as substrate, Zhao et al.

(2006, 2007) introduced four genes into E. coli to produce single chromophore

β-subunit of C-phycocyanin from M. laminosus with spectroscopic properties

characteristic of the native single chromophore β-subunit. Besides the genes for the

apoprotein (cpcB, cpcB(C84S), cpcB(C155I)) and the lyase (CpcS or cpcT) from

Anabaena PCC7120, these included the two respective genes for PCB synthesis, viz.

ho1 and pcyA, from Anabaena PCC7120 (Fig.3-27)

β -phycocyanin-C84,155-PCB2

Cys CysS-PCB S-PCB

84 155

CpcS

CpcT

HO1

PcyACpcS

+

β -phycocyanin-C155-PCB

CysCys

SH SH

Apo-β -phycocyanin 3Z-phycocyanobilin

Biliverdin IX αHeme

β -phycocyanin-C84-PCB

NN N

COO-COO-

N O

SCys

H

HOH

HH

Cys

NN N

COO-COO-

N O

SCys

H

HOH

HH Cys

NH

N HN

COOHCOOH

HN

OOH

A

BC

D

1 2

34

5

6

1819

17

16

15

14

31

7

89

1011

12

13

NH

N N

COOHCOOH

HN

OO

H

D

C B

A

Fig. 3-27: Minimal biosynthetic pathway for the production of PCB from heme and addition to individual binding sites of the C-phycocyanin apo-β subunit.


3.4.2 Expression and Purification of the β-subunits of C-phycocyanin

The plasmids pCDFDuet-cpcS or pETDuet-cpcT, pACYCDuet-ho1-pcyA and

pET-cpcB (or cpcB(C84S), cpcB(C155I)) were introduced in the E. coli strain

BL21(DE3). Upon induction with IPTG, the culture acquired a pronounced blue

tint. The various His-tagged subunits of C-phycocyanin were purified by affinity

chromatography. The absorption and fluorescence emission properties of the

dialyzed chromoproteins are shown in. Fig. 3-28 and Tab. 3-11.

0

30

60

90

120

150

180

Fluo

resc

ence

[a.u

.]300 400 500 600 700 800

0.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

50

100

150

200

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 800

0.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

50

100

150

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

50

100

150

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

A B

C D

FIG. 3-28: Absorption (solid) and fluorescence emission (dashed) spectra of M. laminosus CpcB-C84-PCB(A), CpcB(C155I)-C84-PCB(B), CpcB-C155-PCB(C) and CpcB(C84S)-C155-PCB(D) expressed in E. coli.

The absorption spectrum of HT-CpcB-C84-PCB had a λmax at 619 nm and the bright

red fluorescence had a λmax at 643 nm (Fig. 3-28A). Identical results were obtained

with a mutant lacking the β-155 binding site, namely CpcB(C155I) (Fig. 3-28B). When

pHO1-PcyA and pETDuet-CpcT(All5339) were co-transformed into E. coli BL21(DE3),

together with pET-CpcB(C84S) or pET-CpcB, the biosynthesized and purified


phycobiliprotein had an absorption maximum at 595 nm and a fluorescence maximum

at 625 nm (Fig.3-28C,D). The affinity-purified products obtained with CpcS have the

intense visible absorption and fluorescence typical of native phycobiliproteins (see

Table 3-11, εVis, QVis/uv and ΦF), with the position of the bands red-shifted compared

with holo-β-CPC, peaking in the range associated with the cysteine-β84 chromophore

(CpcS). The products obtained with CpcT had. Vive versa, blue-shifted maxima as

compared to holo-β-CPC, peaking in the range associated with the cysteine-β155

chromophore of CPC (Debreczeny et al., 1995; Sauer and Scheer 1988). We

therefore conclude that CpcS catalyses the site-selective attachment of PCB to

cysteine-ß84 in CpcB; and CpcT catalyses the site-selective attachment of PCB to

cysteine-ß155 in CpcB.

Table 3-11: Quantitative absorption and fluorescence data of the biosynthesized and purified CpcB-C84-PCB, CpcB(C155I)-C84-PCB, CpcB-C155-PCB and CpcB(C84S)-C155-PCB.


CD spectra of reconstituted and DEAE column purified CpcB-C84-PCB,

CpcB(C155I)-C84-PCB, CpcB-C155-PCB and CpcB(C84S)-C155-PCB are

summarized in Fig. 3-29 and in Tab. 3-12

The CD spectra of all four chromoproteins indicated that the pattern (+ for red band, -

for blue band) and intensities are typical for PCB chromophores in their native

conformations, and the maxima of the red bands show the same red shifts for

C84-PCB compared to C155-PCB as the absoption spectra. However, there are two

bands at 330 nm (+) and 370 nm (-) of the C155-PCB in the near uv, which is different

from C84-PCB. In x-ray structures, C155-PCB has a different overall conformation

and C31 stereochemistry than C84-PCB on both subunits (Dürring et al., 1991; Adir et

al., 2001). The spectrum then probably reflects the unusual stereochemistry, these

Absorption Fluorescence Biliprotein

λmax [nm] (QVis/uv) εVis [M-1•cm-1] λmax [nm] ΦF CpcB-C84-PCB 338/619 (2.6) 132000 644 0.11

CpcB(C155I)-C84-PCB 338/619 (2.5) 122000 644 0.23 CpcB-C155-PCB 338/595 (3.3) 113000 625 0.40

CpcB(C84S)-C155-PCB 337/596 (5.2) 106000 626 0.34


are the first CD spectra of individual C155-PCB in the native state, which do reflect

the stereochemistry. The far-UV CD spectra of all four chromoproteins are typical for

α-helical proteins. This confirms native conformations of the protein.

300 400 500 600 700 800-25

-20

-15

-10

-5

0

5

10

15

20

25

CD [a

.u]

Wavelength [nm]

200 220 240 260 280 300-20

-15

-10

-5

0

5

CD [a

.u]

Wavelength [nm]

300 400 500 600 700 800-6

-3

0

3

6

9

12

CD [a

.u]

Wavelength [nm]

200 220 240 260 280 300-20

-15

-10

-5

0

5

CD [a

.u]

Wavelength [nm]

300 400 500 600 700 800-5

0

5

10

15

CD [a

.u]

Wavelength [nm]

200 220 240 260 280 300-20

-15

-10

-5

0

5

CD [a

.u]

Wavelength [nm]

300 400 500 600 700 800

-20

-10

0

10

20

CD [a

.u]

Wavelength [nm]

200 220 240 260 280 300-15

-10

-5

0

5

CD [a

.u]

Wavelength [nm]

A B

C D

FIG. 3-29: CD spectra of reconstituted and DEAE column purified CpcB-C84-PCB(A), CpcB(C155I)-C84-PCB(B), CpcB-C155-PCB(C) and CpcB(C84S)-C155-PCB(D) CD spectra, Vis- CD (top) and far-UV CD spectra (bottom) of M. laminosus β subunit of C-phycocyanin reconstituted in E. coli, affinity chromatography and DEAE column purified.

Table 3-12: CD bands of CpcB-biliproteins



chromoproteins (CpcB-C84-PCB, CpcB(C155I)-C84-PCB, CpcB-C155-PCB and

CpcB(C84S)-C155-PCB) gave nearly identical spectra with maximal absorption


CpcB-C84-PCB 615 (+) 344 (-) 208/221 CpcB(C155I)-C84-PCB 614 (+) 342 (-) 208/222

CpcB-C155-PCB 607 (+) 377 (-) 208/221 CpcB(C84S)-C155-PCB 606 (+) 368 (-) 208/222


around 660 nm. This is evidence for an intact, cysteine-bound PCB and the absence

of significant amounts of mesobiliverdin, which absorbs at longer wavelengths

(Arciero et al., 1988a,b). Purified PCB-C84-CpcB(C155I) and PCB-C155-CpcB(C84S)

were digested with pepsin under acidic conditions, chromopeptides enriched by


(Storf, 2003). The chromopeptides absorb maximally around 660 nm (in acid

condition) (Fig.3-31), which is characteristic for PCB-chromopeptides (Arciero et al.,

1988; Zhao et al., 2006). HPLC analyses gave in both cases one major and several

minor peaks (Fig. 3-30), which were at identical positions to the respective ones

arising from CpcB isolated from the parent cyanobacterium, M. laminosus. This

further supports that CpcS catalyses the site-selective attachment of PCB to

cysteine-ß84 in CpcB; and CpcT catalyses the site-selective attachment of PCB to

cysteine-ß155 in CpcB.

0 . 0 0

0 . 0 5

0 . 1 0

0 . 1 5

0 . 0 0

0 . 0 5

0 . 1 0

0 . 1 5

0 . 0 0

0 . 0 5

0 . 1 0

0 . 1 5

1 0 2 0 3 0 4 00 . 0 0

0 . 0 5

0 . 1 0

0 . 1 5

12 3

1

2 3

Abs

orpt

ion

[a.u

.]

R e t e n t io n t im e [ m in ]

1 23

FIG. 3-30: HPLC (λdetect = 650 nm) of the chromopeptides obtained from peptic digestion of the sequentially reconstituted CpcB-C84,C155-PCB2 (data from Zhao et al., 2007b) (top), CpcB(C84S)-C155-PCB(second), CpcB(C155I)-C84-PCB (third) and β-CPC (bottom). HPLC were eluted using a gradient (80:20 to 60:40) of formic acid (0.1%, pH 2) and acetonitrile containing 0.1% formic acid. CpcB from M. laminosus.


300 400 500 600 700 8000.00

0.04

0.08

0.12

0.16

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

C

300 400 500 600 700 800

0.04

0.08

0.12

0.16

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

A

300 400 500 600 700 8000.00

0.04

0.08

0.12

0.16

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

B

FIG. 3-31: Absorption spectra of major peptic chromopeptide peaks in the chromatograms shown in Fig. 3-30. A: Peak 1 derived from the sequentially reconstituted CpcB-C84,155-PCB2 (Fig.3-31, top), from singly-chromophorylated CpcB(C1551) (Fig. 3-31, second), and β-CPC (Fig. 3-31, bottom). B: Peak 2 derived from the sequentially-reconstituted CpcB-C84,155-PCB2 (Fig.3-31, top), from singly chromophorylated CpcB(C84S) (Fig. 3-31, third), and β-CPC (Fig. 3-31, bottom). C: sequentially reconstituted CpcB-C84,155-PCB2 (Fig.3-31, top), from singly chromophorylated CpcB(C84S) (Fig. 3-31, third), and β-CPC (Fig. 3-31, bottom).


β-subunits of C-phycocyanin

RP-HPLC separation of trypsin digests (Fig.3-32) on a C18 column, gave two types of

colored components. Component 1 (A:tr=17.7 min ; B: tr=17.7 min; C: tr=20.9 min; D:

tr=20.9 min) had an absorption maximum around 640nm (Fig.3-33) that is typical for

PCB-containing peptides; component 2 (A:tr=29.2 min ; B: tr=29.6 min) corresponds to

free PCB according to its retention time and the absorption maximum about at 685

nm (Fig.3-33). We found an interesting phenomenon that there is no free PCB in the

C155-PCB samples, maybe due to PCB tighter binding of PCB to C155 than to C84.


The underlined fractions were used for MS.

10 20 30 400.0

0.1

0.2

0.3

0.4

Abs

orpt

ion

[a.u

.]


10 20 30 400.0

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orpt

ion

[a.u

.]


10 20 30 40

0.0

0.1

0.2

0.3

Abs

orpt

ion

[a.u

.]


1

2

10 20 30 400.0

0.1

0.2

0.3

0.4

Abs

orpt

ion

[a.u

.]


1 2B

A

D

C

FIG. 3-32: HPLC(λdetect = 650 nm) of the chromopeptides obtained from tryptic digestion of the reconstituted β-subunit of C-phycocyanin (CpcB-C84-PCB(A), CpcB(C155I)-C84-PCB(B), CpcB-C155-PCB(C) and CpcB(C84S)-C155-PCB(D)). Spectra of individual peaks of the chromatograms are shown in Fig. 3-33. HPLC were eluted using a gradient (80:20 to 60:40) of formic acid (0.1%, pH 2) and acetonitrile containing 0.1% formic acid.

300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4 peak 1 peak 2

Abs

orpt

ion

[a.u

]

Wavelength [nm]300 400 500 600 700 800

0.0

0.2

0.4

0.6

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

300 400 500 600 700 8000.0

0.1

0.2

0.3 peak 1 peak 2

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

A

DB

C

FIG. 3-33: Absorption spectra of the chromopeptides obtained from tryptic digestion of the reconstituted β subunit of C-phycocyanin (CpcB-C84-PCB(A), CpcB(C155I)-C84-PCB(B), CpcB-C155-PCB(C) and CpcB(C84S)-C155-PCB(D))..


Sequences of chromophore-containing peptides were deduced from sequences

around the chromophore-binding sites in β-subunits of phycocyanin from M.

laminosus. In native CPC subunits, PCB is bound to cysteine resides 82 and 153 of

the beta subunit. There is excellent agreement between the measured peptide Mr

values and those calculated from sequences of β-subunits of phycocyanin from M.

laminosus (Tab. 3-13). The chromopeptide sequences confirm the attachment of PCB

to the known binding sites (C84 and C155, consensus sequence) of CpcB.

Table 3-13: Mass of chromopeptides from tryptic digestion

Peptide peak (m/z)Biliprotein Chromopeptide Calc. Expt.

CpcB-C84-PCB M(79)AAC(PCB)LR [M+2H]2+ 625.57 625.80 CpcB(C155I)-C84-PCB M(79)AAC(PCB)LR [M+2H]2+ 625.57 625.74

CpcB-C155-PCB G(151)DC(PCB)SAL [M+2H]2+ 575.95 576.19 CpcB(C84S)-C155-PCB G(151)DC(PCB)SAL [M+2H]2+ 575.95 576.19

3.4.6 NMR analysis of peptic chromopeptides from reconstituted β

subunits of C-phycocyanin

RP-HPLC separation of pepsin digests (Fig.3-34) on a C18 column, gave one type of

colored components, including A: tr=18.5, 22.9, 23.6 min; B: tr =18.4, 22.9, 23.6 min;

C: tr =20.3 min and 22.7 min; D: tr =20.4 min and 22.8 min, had an absorption

maximum about at 640nm that is typical for PCB-containing peptides. The underlined

fractions were used to measure NMR spectroscopy.


the 600-MHz 1H NMR spectra of the chromopeptide of β subunit of phycocyanin

(CpcB-C84-PCB and CpcB-C155-PCB) in 10 mM TFA/D2O, were compared (Tab.

3-14 and Fig. 3-36).


10 20 30 400.0

0.1

0.2

0.3

0.4

0.5

Abs

orpt

ion

[a.u

.]


1

2

10 20 30 400.0

0.1

0.2

0.3

0.4

Abs

orpt

ion

[a.u

.]


1

2

10 20 30 400.0

0.2

0.4

0.6

Abs

orpt

ion

[a.u

]


1

2

3

4

10 20 30 400.0

0.2

0.4

0.6

Abs

orpt

ion

[a.u

.]


1

2

3

4

A

B D

C

FIG. 3-34: HPLC(λdetect = 650 nm) of the chromopeptides obtained from peptic digestion of the reconstituted β-subunit of C-phycocyanin (CpcB-C84-PCB(A), CpcB(C155I)-C84-PCB(B), CpcB-C155-PCB(C) and CpcB(C84S)-C155-PCB(D)).

8 7 6 5 4 3 2 1 0Chemical shift [ppm]


A

C

FIG. 3-36: 1-D 1H NMR spectra of peptic chromopeptides from the β subunit of C-phycocyanin (CpcB-C84-PCB(A), CpcB-C155-PCB(C)) in 10 mM TFA/D2O at 25 °C


signals. Parallel analysis of the NMR spectra of β subunit of C-phycocyanin


CpcB-C84-PCB and CpcB-C155-PCB chromopeptides reveals the expected

resonance frequencies of the chromophore (Tab. 3-14). The 1H NMR spectral

assignments of the bilin moiety of chromopeptides show great similarity with those

reported for the blue pigment (Bishop et al., 1986, chapter 3.2 and 3.3). The

assignment of the remaining resonances to protein can be made.

In the downfield region of the spectrum, the three vinylic 10-H, 15-H and 5-H

resonances at 6.91(6.97), 6.26(6.26) and 5.83(5.70) ppm, respectively. The 31-H

multiplet at 3.49(3.50) ppm is downfield shifted due to its proximity to the sulfur atom,


yielding a doublet at 1.38(1.32) ppm. Similarly the 2- CH3 signal (1.14(1.13) ppm) is

coupled to the 2-H proton (2.52(2.53) ppm). The 3-H proton (multiplet at 2.94(2.97)

ppm) is coupled to both the 2-H and 31-H. All these signals are again characteristic of

a PCB bound to cysteine at C-31. We found that there are only very small differences

between the signals of the C84-PCB and C155-PCB chromopeptides, in spite of the

different stereochemistry at C-31. The only distinct difference is for the 5-H, which may

be diagnostic.


group at C- 18, and the propionic acid methylenes at C-8 and -12 are salient features

of the spectra of bile pigments. The three aromatic methyls at C-7, -13, and -17 give

singlets at 2.11(2.10), 2.08(2.01) and 1.99(1.97) ppm. The ethyl group at C-18, is

seen as two multiplets at 1.96(1.95) ppm (18- CH2CH3) and 0.98(0.97) ppm

(18-CH2CH3). The multiplet resonances from methylene group of the propionic acid

side chains at C-8 and C-12 were found around 2.85(2.87) and 2.35(2.36) ppm.

These results correspond well with the A-ring-linked PCB structure (chapter 3.2 and

3.3).

Analysis of the NMR spectra of CpcB-C84-PCB and CpcB-C155-PCB

chromopeptides reveals in both cases the resonances of the expected amino acid

residues (Table 3-14). All amino acid α protons appear between 3.8 and 4.6 ppm. The

chemical shifts of the α hydrogens of these same amino acids (Ala, Cys, Thr) in

CpcB-C84-PCB chromopeptide are displaced within 0.03 ppm upfield from those in

the CpcB-C155-PCB chromopeptide. Chemical shifts are not observed for some of

the resonances of the δ protons of Lys and Arg; and the γ proton of Met of


chromopeptide. Major differences in the spectra of the two chromopeptides are

evident in the resonances attributed to the Arg, Asn, Asp, Gly, Leu, Lys, Met and Ser

residues. Based on the sequences around the chromophore-binding sites in

CpcB-C84-PCB and CpcB-C155-PCB from M. laminosus, chromopeptides sequence

TNRRMAAC(PCB) of CpcB-C84-PCB and TKGDC(PCB)SAL of CpcB-C155-PCB

were deduced. These confirm the attachment of PCB to correct site of CpcB by CpcS

and CpcT lyase catalysis.

Table 3-14: 1H NMR (600-MHz) assignments for peptide TNRRMAAC(PCB) of reconstituted CpcB-C84-PCB and peptide TKGDC(PCB)SAL of reconstituted CpcB-C155-PCB ( 10 mM TFA/D2O)

Chemical shift CpcB-C84-PCB CpcB-C155-PCB

Multiplicity (JH-H,[Hz])


6.91 6.97 s 1 10-H 6.26 6.26 s 1 15-H 5.83 5.70 s 1 5-H 3.49 3.50 m 1 3’-H 2.94 2.97 m 1 3-H 2.85 2.87 m 4 8,12-CH2CH2COOH 2.52 2.53 m 1 2-H 2.35 2.36 m 4 8,12-CH2CH2COOH 2.11 2.10 s 2.08 2.01 s 1.99 1.97 s

9 7,13,17-CH3

1.96 1.95 m 2 18- CH2CH3 1.38 1.32 d 3 3’- CH3 1.14 1.13 d 3 2-CH3 0.98 0.97 m 3 18- CH2CH3

4.05, 4.14 4.04 q 1 Ala α- CH 1.20 1.27 d 3 Ala β- CH3

4,12, 4.05 / m 1 Arg α- CH 1.51 / d 2 Arg β- CH2 1.27 / m 2 Arg γ- CH2

/ / / 2 Arg δ- CH2 4.36 / m 1 Asn α- CH 2.86 / m 2 Asn β- CH2

/ / / 1 Asp α- CH / 2.87 m 2 Asp β- CH2

4.38 4.38 m 1 Cys α- CH 3.00 3.15 m 2 Cys β- CH2

/ 3.87 d 1 Gly α- CH


/ 4.42 d 1 Leu α- CH / / / 2 Leu β- CH2 / 0.89 d 1 Leu γ- CH / 0.85 d 2 Leu δ-CH2 / 4.55 m 1 Lys α- CH / 1.53 d 2 Lys β- CH2 / 1.47 m 2 Lys γ- CH2 / / 2 Lys δ- CH2

4.16 / m 1 Met α- CH 2.18 / m 2 Met β- CH2

/ / / 2 Met γ- CH2 / 4.32 m 1 Ser α- CH / 3.83 m 2 Ser β- CH2

4.14 4.11 t 1 Thr α- CH 3.58 3.59 d 2 Thr β- CH2 1.27 1.27 d 3 Thr γ- CH3

3.4.7 Discussion

The studies described here documented successful reconstitution of PCB to β-subunit

of CPC, CpcB, and thereby the proposed pathway for the biosynthesis of the

phycocyanin β subunit (Fig.3-27) in E. coli. The results of experiments support and

extend the conclusion reached by Shen et al. (2006, 2008), Saunee et al. (2008) and

Zhao et al. (2006a, 2007b) that the lyases CpcS and CpcT catalyse the site-selective

attachment of PCB to cysteine-β84 and 155, respectively, in CpcB.

Compared with enzymatic chromophore attachment to the α-subunits, relatively little

has been known, until recently, about the catalytic chromophore attachment to the

β-subunits of phycobiliproteins, where the process is complicated by the presence of

two or even more binding sites (Glazer, 1994; Grossman et al., 1993; Zhao et al.,

2007; Sidler, 1994). Nonenzymatic attachment of PCB to CpcB from M. laminosus

indicated that both binding sites can bind the chromophore spontaneously, and that

the site-specificity can be influenced by the reaction medium: binding of C155 is

favored under the influence of the detergent, Triton X- 100, and binding to C84 in its

absence (Zhao et al., 2004). The spontaneous reaction, however, is unspecific.

Moreover, experiments with multiply transformed E. coli indicate that a spontaneous

addition to either site is very low in vivo, at least in E. coli (Zhao et al., 2006, 2007b).


The identification of a set of four genes (cpcS, T, U and V) from Synechococcus sp.

PCC7002 that catalyze the addition of PCB to phycobiliprotein β-subunits (Shen et al.,

2004) initiated a better understanding of the process. Distinct specificities for the two

binding sites of β-CPC recently became obvious for these proteins. Under catalysis of

CpcS-I-CpcU, from Synechococcus PCC7002, PCB is attached regioselectively to

cysteine-β84 (Shen et al., 2008; Saunee et al., 2008). Conversely, CpcT from

Synechococcus PCC7002 catalyzed the attachment to cysteine-155 of CpcB (Shen et

al., 2006). The site specificity of CpcS and CpcT are fully supported by our finding that

the homologous CpcS and CpcT from Anabaena PCC7120, catalyzes the attachment

of PCB to cysteine-β84 and cysteine-β155 of CpcB. It is noteworthy that most of the

work reported here was done with CpcB from M. laminosus; and the lyase was from

Anabaena PCC7120 CpcT catalyzes equally well the PCB attachment to CpcB from

Anabaena PCC7120 (Zhao et al., 2007b). A similar cross-reactivity was shown for the

cysteine-84 lyase, CpcS (Zhao et al., 2006a). Taken together, chromophore

attachment to the β-subunit of CpcB is catalyzed by enzymes that are specific for the

two binding sites, Cys-β84 and Cys-β155, but both lyases have a broader specificity

to their substrate proteins than the α84 lyases, CpcE/F and PecE/F.

The lyase CpcS (CpcT) catalyses the site-selective attachment of PCB to

cysteine-β84(155) in CpcB, resulting in CpcB-C84-PCB (CpcB-C155-PCB), as

reported in Zhao et al. (2007b), they tested whether by combined catalysis of CpcT

and CpcS, holo-β-CPC could be generated in the E. coli system. The results showed

that the purified phycobiliprotein formed had an absorption maximum at 618 nm,

which is red-shifted from that of of β-CPC isolated from M.laminosus (λmax = 609 nm).

By contrast,however, the fluorescence maxima were the same for the reconstituted

chromoprotein and the isolated subunit (644 nm). The PCB-C155 and PCB-C84

chromopeptide ratio of 0.7:1 was estimated of from HPLC analysis of the

chromopeptides (Fig. 3-30, sequentially reconstituted CpcB-C84,155-PCB2). In

β-CPC, PCB-C84 absorbs at longer wavelength than PCB-C155 and is the

predominantly fluorescing chromophore. The data thus indicate the correct

attachment of PCB to C84 when the C155-PCB is already present, but an incomplete

or incorrect reconstitution of PCB to C155 when the C84-PCB is already present. In

part, the failure to obtain doubly chromophorylated proteins when both lyases are

present, could relate to the relatively low kcat of CpcT as compared to CpcS. This is an


interesting problem which indicates that unknown regulatory factors may be present in

cyanobacteria.

In contrast to Synechococcus PCC7002, two copies each of cpeS- and cpeT-like

genes are present in Anabaena PCC7120. Only CpcT1 (all5339) is capable of

correctly attaching PCB to cysteine-155 of CpcB, while its homologue CpcT2 is

inactive in vitro and in E. coli. Similarly, only CpcS1 (alr0617) catalyzes the

attachment of PCB to cysteine-84 of CpcB, while its homologue, CpcS2, is inactive. It

should be noted that the kinetic constants of CpcT1 is lower than those of CpcS1 and

the EF-type lyases.(Zhao et al., 2007b)

The evolution of site-specific enzymes may relate to the different binding situations of

the two chromophores on the β-subunits. The conformation of PCB bound to

cysteine-β155 differs from that of PCB bound to cysteines β84 (and α84) (Schirmer et

al., 1987), as is the stereochemistry of the asymmetric C-31 of PCB that is generated,

together with the asymmetric C-3, during the addition reaction. In the high-resolution

structures, C-31 is R-configured at PCB bound to cysteine-84, whereas it is

S-configured in PCB bound to cysteine-155 (Ritter et al., 1999; Wang et al., 2001; Adir

et al., 2002). Conceivably, this different binding requires a different conformations of

the chromophore when it is presented by the lyase to the binding site, as indicated by

a conformation-dependent site selectivity of the autocatalytic addition reaction (Zhao

et al., 2004).

The identification and characterization of the CpcS and CpcT lyases in several

organisms, and of their interactions, and also the possibility for the coexpression of

the various components in E. coli now opens a new approach to study the assembly

of phycobilisomes, the structures responsible for a major fraction of global

photosynthesis.


3.5 Structure analysis of reconstituted β-subunits of

phycoerythrocyanin

3.5.1 Reconstitution principle of β-subunit of phycoerythrocyanin

Phycocyanobilin (PCB) is biosynthesized from heme by the action of two enzymes,

heme oxygenase and BV-reductase. Similar to the reaction with CpcB (section 3.4),

PCB is then attached to the β subunit apoprotein of phycoerythrocyanin by a lyase,

CpcS (Zhao et al. 2006) or CpcT (Zhao et al. 2007; Shen et al. 2006). Using

endogenous heme as substrate, Zhao et al. (2006, 2007) introduced four genes into E.

coli to produce single-chromophore β-subunits of phycoerythrocyanin from M.

laminosus with spectroscopic properties characteristic of the individual chromophores

in the native β-subunit. Besides the genes for the apoprotein (pecb, pecb(C84A),

pecB(C155I)) and the lyase (CpcS or cpcT) from Anabaena PCC7120, these included

the two respective genes for PCB synthesis, viz. ho1 and pcyA, from Anabaena

PCC7120. The reconstitution principle of the β-subunit of phycoerythrocyanin is

similar to tha of β-CPC (see Fig. 3-27).

3.5.2 Expression and Purification of the β-subunits of

phycoerythrocyanin

Expression followed the principle for CpcB (section 3.4.2), using pCDFDuet-cpcS or

pETDuet-cpcT, pACYCDuet-ho1-pcyA and pET-pecB (or pecB(C84S), pecB(C155I))

plasmids in the E. coli strain BL21(DE3). Upon induction with IPTG, the culture

acquired a pronounced blue tint. The various His-tagged subunits of

phycoerythrocyanin were purified by affinity chromatography. The absorption and

fluorescence emission properties of the dialyzed chromoproteins are shown in. Fig.

3-37 and Tab. 3-16.


0

100

200

300

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

100

200

300

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

50

100

150

200

250

300

350

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

80

160

240

320

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

A B

C D

FIG. 3-37: Absorption (solid) and fluorescence emission (dashed) spectra of M. laminosus PecB-C84-PCB(A), PecB(C155I)-C84-PCB(B), PecB-C155-PCB(C) and PecB(C84A)-C155-PCB(D) expressed in E. coli.

The absorption spectrum of HT-PecB-C84-PCB had a λmax at 602 nm and the bright

red fluorescence had a λmax at 629 nm (Fig. 3-37A). Identical results were obtained

with a mutant lacking the β-155 binding site, namely PecB(C155I) (Fig. 3-37B).

When pHO1-PcyA and pETDuet-CpcT were co-transformed into E. coli BL21(DE3),

together with pET-PecB(C84A) or pET-PecB, the biosynthesized and purified

phycobiliprotein had an absorption at 596 nm and fluorescence at 625 nm (Fig.3-37

C,D). The affinity-purified products obtained with CpcS have the intense visible

absorption and fluorescence typical of native phycobiliproteins (see Table 3-11, εVis,

QVis/uv and ΦF), with the position of the bands red-shifted compared with holo-β-PEC,

peaking in the range associated with the cysteine-β84 chromophore (CpcS). The

products obtained with CpcT versa, blue-shifted maxima as compared to holo-β-PEC,

peaking in the range associated with the cysteine-β155 chromophore of PEC from

M. laminosus (Parbel et al., 1997). We therefore conclude that CpcS catalyses the

site-selective attachment of PCB to cysteine-ß84 in PecB; and CpcT catalyses the

site-selective attachment of PCB to cysteine-β155 in PecB.


Table 3-16: Quantitative absorption and fluorescence data of the biosynthesised and purified PecB-C84-PCB, PecB(C155I)-C84-PCB, PecB-C155-PCB and PecB(C84A)-C155-PCB.


The CD spectra (Fig. 3-38 and in Tab. 3-17) of PecB-C84-PCB,

PecB(C155I)-C84-PCB, PecB-C155-PCB and PecB(C84A)-C155-PCB are typical for

PCB-containing biliproteins. But the CD signals of PecB-C155-PCB and

PecB(C84A)-C155-PCB are of opposite sign, and the near uv signals of C155-PCB of

PecB are differenct from that of CpcB, reflecting a different chromophore geometry.

This is again the first time that spectra (λmax=596 nm) for the native C155-PCB are

obtained directly, they are somewhat red-shiftd compared to the indirectly obtained

data of Parbel et al. (1991) (λmax=583nm) and the theoretical spectra (λmax=585nm) of

Scharnagl et al. (1991). The absorption maximum of the C155-PCB is red-shifted by

11 nm as compared to the CD-maximum, indicating some microheterogeneity

(Scharnagl et al., 1991). The far-UV CD spectra of all four chromoproteins are typical

for α-helical proteins. This confirms native conformations of both the chromophore

and the protein.

Table 3-17: CD bands of PecB-biliproteins

Absorption Fluorescence Biliprotein λmax [nm] (QVis/uv) εVis [M-1•cm-1] λmax [nm] ΦF

PecB-C84-PCB 337/602 (3.0) 101000 629 0.23 PecB(C155I)-C84-PCB 338/602 (2.6) 116000 629 0.51

PecB-C155-PCB 338/596 (2.9) 108000 625 0.25 PecB(C84A)-C155-PCB 338/596 (2.9) 103000 625 0.34


PecB-C84-PCB 600 (+) 342 (-) 209/222 PecB(C155I)-C84-PCB 599 (+) 342 (-) 209/220

PecB-C155-PCB 595 (-) 336 (+) 209/222 PecB(C84A)-C155-PCB 597 (-) 335 (+) 209/222


200 220 240 260 280 300-20

-15

-10

-5

0

5

CD [a

.u]

Wavelength [nm]

300 400 500 600 700 800-5

0

5

10

15

20

25

CD [a

.u]

Wavelength [nm]

200 220 240 260 280 300-20

-15

-10

-5

0

5

CD [a

.u]

Wavelength [nm]

300 400 500 600 700 800-10

-5

0

5

10

15

20

25

30

CD [a

.u]

Wavelength [nm]

200 220 240 260 280 300

-10

-5

0

CD [a

.u]

Wavelength [nm]

300 400 500 600 700 800

-10

-5

0

5

10

15

CD [a

.u]

Wavelength [nm]

200 220 240 260 280 300-15

-10

-5

0

5

CD [a

.u]

Wavelength [nm]

300 400 500 600 700 800-10

-5

0

5

10

CD [a

.u]

Wavelength [nm]

A B

C D

FIG. 3-38: CD spectra of reconstituted and DEAE column purified PecB-C84-PCB(A), PecB(C155I)-C84-PCB(B), PecB-C155-PCB(C) and PecB(C84A)-C155-PCB(D) CD spectra, Vis- CD (top) and far-UV CD spectra (bottom) of M. laminosus β subunit of C-phycocyanin reconstituted in E. coli, affinity chromatography and DEAE column purified.



chromoproteins (PecB-C84-PCB, PecB(C155I)-C84-PCB, PecB-C155-PCB and

PecB(C84A)-C155-PCB) gave nearly identical spectra with maximal absorption

around 660 nm. This is evidence for an intact, cysteine-bound PCB and the absence

of significant amounts of mesobiliverdin, which absorbs at longer wavelengths

(Arciero et al., 1988a,b). Purified PecB(C155I)-C84-PCB and PecB(C84A)-C155-PCB

were digested with pepsin under acidic conditions, chromopeptides enriched by


(Storf, 2003). The chromopeptides absorb maximally around 660 nm (in acid

condition) (Fig.3-40), which is characteristic for PCB-chromopeptides (Arciero et al.,

1988b; Zhao et al., 2006). HPLC analyses gave in both cases one major and several

minor peaks (Fig. 3-39), which were at identical positions to the respective ones

arising from PEC isolated from the parent cyanobacterium, M. laminosus. In summary,


therefore, we conclude that CpcS catalyses the site-selective attachment of PCB to

cysteine-ß84 in PecB; and CpcT catalyses the site-selective attachment of PCB to

cysteine-ß155 in PecB.

10 20 30 400.000.050.100.150.000.050.100.150.000.050.100.150.000.050.100.15

21

R etention tim e [m in]

Abs

orpt

ion

[a.u

.]

2

1

21

FIG. 3-39: HPLC (λdetect = 650 nm) of the chromopeptides obtained from peptic digestion of the sequentially reconstituted PecB-C84,C155-PCB2 (data from Zhao et al., 2007b) (top), PecB(C155I)-C84-PCB (second), PecB(C84A)-C155-PCB (third) and β-PEC (bottom). HPLC were eluted using a gradient (80:20 to 60:40) of formic acid (0.1%, pH 2) and acetonitrile containing 0.1% formic acid.

300 400 500 600 700 800

0.04

0.08

0.12

0.16

Abso

rptio

n [a

.u.]

Wavelength [nm]

B

300 400 500 600 700 8000.00

0.04

0.08

0.12

0.16

Abso

rptio

n [a

.u.]

Wavelength [nm]

A

FIG. 3-40: Absorption spectra of major peptic chromopeptide peaks in the chromatograms shown in Fig.3-39. A: Peak 1 derived from the sequentially reconstituted PecB-C84,155-PCB2 (Fig.3-39, top), from singly-chromophorylated PecB(C1551) (Fig. 3-39, second), and β-PEC (Fig. 3-39, bottom). B: Peak 2 derived from the sequentially-reconstituted PecB-C84,155- PCB2 (Fig.3-39, top), from singly chromophorylated PecB(C84A) (Fig. 3-39, third), and β-PEC (Fig. 3-39, bottom).



β-subunits of phycoerythrocyanin

RP-HPLC separation of trypsin digests (Fig.3-41) on a C18 column, gave two

types of colored components. Component 1 (A:tr =14.2 min ; B: tr =14.0 min; C:

tr =19.4, 25.1, 26.2 and 30.8 min; D: tr =19.7, 25.1, 26.4, 30.9 min) had an

absorption maximum around 645nm (Fig.3-23) that is typical for

PCB-containing peptides; component 2 (A:tr = 29.5 min ; B: tr = 28.2 min)

corresponds to free PCB according to its retention time and the absorption

maximum about at 685 nm (Fig.3- 42). The underlined fractions were used for

MS.

10 20 30 400.00

0.05

0.10

0.15

0.20

0.25

Abs

orpt

ion

[a.u

.]


1 2 3

4

10 20 30 400.00

0.05

0.10

0.15

0.20

Abs

orpt

ion

[a.u

.]


12 3

4

10 20 30 400.0

0.1

0.2

0.3

0.4

Abs

orpt

ion

[a.u

.]


1

2

10 20 30 400.0

0.1

0.2

0.3

Abs

orpt

ion

[a.u

.]


1

2

A

B

C

D

FIG. 3-41: HPLC(λdetect = 650 nm) of the chromopeptides obtained from tryptic digestion of the reconstituted β-subunit of phycoerythrocyanin (PecB-C84-PCB(A), PecB(C155I)-C84-PCB(B), PecB-C155-PCB(C) and PecB(C84A)-C155-PCB(D)). Spectra of individual peaks of the chromatograms are shown in Fig. 3-43. HPLC were eluted using a gradient (80:20 to 60:40) of formic acid (0.1%, pH 2) and acetonitrile containing 0.1% formic acid.


300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

0.25

Wavelength [nm]

Abs

orpt

ion

[a.u

.]


300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

0.25

Wavelength [nm]

Abs

orpt

ion

[a.u

.]


300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

Wavelength [nm]

Abs

orpt

ion

[a.u

.]

peak 1 peak 2

300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

Wavelength [nm]

Abs

orpt

ion

[a.u

.] peak 1 peak 2A

B

C

D

FIG. 3-42: Absorption spectra of the chromopeptides obtained from tryptic digestion of the reconstituted β subunits of phycoerythrocyanin (PecB-C84-PCB(A), PecB(C155I)-C84-PCB(B), PecB-C155-PCB(C) and PecB(C84A)-C155-PCB(D)).

Sequences of chromophore-containing peptides were deduced from

sequences around the chromophore-binding sites in β-subunits of

phycoerythrocyanin from M. laminosus. In native PEC subunits, PCB is bound

to cysteine resides 82 and 153 of the β-subunit. There is excellent agreement between the measured peptide Mr values and those values calculated from

sequences of beta subunits of phycoerythrocyanin from M. laminosus (Table

3-18). The chromopeptide sequences confirm the attachment of PCB to the

known binding sites (C84 and C155, consensus sequence) of PecB.

Table 3-18 Mass of chromopeptides from tryptic digestion

Peptide peak (m/z) Biliprotein Chromopeptide Calc. Expt.

PecB-C84-PCB N(78)QAAC(PCB)IR [M+2H]2+ 681.09 681.26 PecB(C155I)-C84-PCB N(78)QAAC(PCB)IR [M+2H]2+ 681.09 681.26

PecB-C155-PCB G(151)DC(PCB)SQLMSEL [M+2H]2+ 834.75 834.75

PecB(C84A)-C155-PCB G(151)DC(PCB)SQLMSELAS

YFDR [M+2H]2+ 1204.64 1204.42


3.5.6 NMR analysis of peptic chromopeptides from reconstituted

β-subunits of phycoerythrocyanin

RP-HPLC separation of pepsin digests (Fig.3-43) on a C18 column gave one type of

colored components, including A:tr=17.1 min and tr=21.2 min ; B: tr=16.8 min and

tr=21.0 min; C: tr=18.9min and tr=20.9 min ; D: tr=19.1min and tr=21.1 min, they all had

an absorption maximum about at 640nm that is typical for PCB-containing peptides in

the solvent system used. The underlined fractions were used to measure NMR

spectroscopy.


the 600-MHz 1H NMR spectra of the chromopeptides of PEC β-subunits

(PecB-C84-PCB and PecB(C84A)-C155-PCB) in 10 mM TFA/D2O, were compared

(Table 3-19 and Fig. 3-45)

.

10 20 30 400.0

0.2

0.4

0.6

0.8

Abs

orpt

ion

[a.u

.]


1

2

10 20 30 400.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


Abs

orpt

ion

[a.u

.]

1

2

10 20 30 400.0

0.2

0.4

0.6

0.8

1.0


Abs

orpt

ion

[a.u

.]

1

2

10 20 30 400.0

0.2

0.4

0.6

0.8


Abs

orpt

ion

[a.u

.]

1

2

A

B

C

D

FIG. 3-43: HPLC(λdetect = 650 nm) of the chromopeptides obtained from peptic digestion of the reconstituted β-subunits of phycoerythrocyanin (PecB-C84-PCB(A), PecB(C155I)-C84-PCB(B), PecB-C155-PCB(C) and PecB(C84A)-C155-PCB(D)).


8 7 6 5 4 3 2 1 0


8 7 6 5 4 3 2 1 0


A

D

FIG. 3-45: 1-D 1H NMR spectra of chromopeptides of PEC β subunits (PecB-C84-PCB(A), PecB(C84A)-C155-PCB(D)) in 10 mM TFA/D2O at 25 °C


signals. Parallel analysis of the NMR spectra of β subunit of phycoerythrocyanin

PecB-C84-PCB(PecB(C84A)-C155-PCB) chromopeptide reveals the expected

similarities of the resonance frequencies of the chromophore (Table 3-19). The 1H

NMR spectral assignments of the bilin moiety of chromopeptides show great similarity

with those reported for the blue PEC-derived pigment (Bishop et al., 1986, section 3.2,

3.3 and 3.4). The assignment of the remaining resonances to protein can be made.

In the downfield region of the spectrum, there are three vinylic 10-H, 15-H and 5-H

resonances at 7.01(7.16), 6.26(6.22) and 5.74(5.66) ppm, respectively. The 31-H

multiplet at 3.51(3.51) ppm is downfield shifted due to its proximity to the sulfur atom,


yielding a doublet at 1.33(1.28) ppm. Similarly the 2- CH3 proton (1.13 ppm) is coupled

to the 2-H proton (2.53(2.67) ppm). The 3-H proton (multiplet at 2.96(2.98) ppm) is

coupled to both the 2-H and 31-H. All these signals are again characteristic of a PCB

bound to cysteine at C-31. We found that there are only very small differences

between the signals of the C84-PCB and C155-PCB chromopeptides, in spite of the


different stereochemistry at C-31. The only distinct difference is for the 10-H and 5-H,

which may be diagnostic.

Table 3-19: 1H NMR (600-MHz) assignments for peptide HHRNQAAC(PCB)IRD of reconstituted PecB-C84-PCB and peptide TKGDC(PCB)SQL of reconstituted PecB(C84A)-C155-PCB (10 mM TFA/D2O).

Chemical shift PecB-C84-PCB PecB(C84A)-C155-PCB


Number of H’s

Assignment

7.01 7.16 s 1 10-H 6.26 6.22 s 1 15-H 5.74 5.66 s 1 5-H 3.51 3.51 m 1 3’-H 2.96 2.98 m 1 3-H 2.87 2.93 m 4 8,12-CH2CH2COOH 2.53 2.67 m 1 2-H 2.40 2.41 m 4 8,12-CH2CH2COOH2.10 2.09 s 2.08 2.07 s 2.01 2.00 s

9 7,13,17-CH3

1.98 1.94 m 2 18- CH2CH3 1.33 1.28 m 3 3’- CH3

/ 1.13 / 3 2-CH3 0.89 0.90 m 3 18- CH2CH3

4.05, 3.90 / q 1 Ala α- CH

1.27 / d 3 Ala β- CH3 4.11, 4.05 / m 1 Arg α- CH

1.53 / m 2 Arg β- CH2 1.27 / d 2 Arg γ- CH2

/ / / 2 Arg δ- CH2 4.32 / m 1 Asn α- CH 2.90 / m 2 Asn β- CH2

/ 4.61 / 1 Asp α- CH 2.63 2.85 m 2 Asp β- CH2 4.32 4.30 m 1 Cys α- CH 3.04 3.13 m 2 Cys β- CH2 4.15 4.16 m 1 Gln α- CH 2.39 2.25 m 2 Gln β- CH2

/ 2.25 / 2 Gln γ- CH2 / 3.91 d 1 Gly α- CH

4.20, 4.15 / m 1 His α- CH 3.16 / m 2 His β- CH2


/ / / 2 His γ- CH2 4.36 / m 1 Ile α- CH 0.83 / m 1 Ile β- CH

/ / / 2 Ile γ- CH2 0.83 / m 3 Ile δ-CH3

/ 4.39 q 1 Leu α- CH / 1.53 m 2 Leu β- CH2 / 0.83 d 1 Leu γ- CH / 0.81 d 2 Leu δ-CH2 / 4.55 m 1 Lys α- CH / 1.54 m 2 Lys β- CH2 / 1.52 d 2 Lys γ- CH2 / / / 2 Lys δ- CH2 / 4.12 m 1 Ser α- CH / 3.83 m 2 Ser β- CH2 / 4.06 m 1 Thr α- CH / 3.60 m 2 Thr β- CH2 / 1.28 d 3 Thr γ- CH3


group at C- 18, and the propionic acid methylenes at C-8 and -1 2 are salient features

of the spectra of bile pigments. The three aromatic methyls at C-7, -13, and -17 show

as singlets at 2.10(2.09), 2.08(2.07) and 2.01(2.00) ppm. The ethyl group at C-18 is

seen as two multiplets at 1.98(1.94) ppm (18- CH2CH3) and 0.89(0.90) ppm

(18-CH2CH3). The multiplet resonances from the methylene group of the propionic

acid side chains at C-8 and C-12 were found at 2.87(2.93) and 2.40(2.41) ppm. These

results correspond well with the A-ring-linked PCB structure (section 3.2, 3.3 and 3.4)

Analysis of the NMR spectra of PecB-C84-PCB and PecB(C84A)-C155-PCB

chromopeptides reveals the resonances of the expected amino acid residues (Table

3-19). All amino acid α-protons appear between 3.8 and 4.6 ppm. The chemical shifts

of the α hydrogens of Gln and Cys in PecB-C84-PCB chromopeptide are ≤ 0.03 ppm

upfield from the respective ones in the PecB(C84A)-C155-PCB chromopeptide. No

signals were observed for some of the resonances of the δ protons of Lys and Arg; γ

protons of Gln, His and Ile of the chromopeptide. The expected resonance of Asp

α-CH was hidden under the resonance of the solvent. Major differences in the spectra

of the two chromopeptides are evident in the resonances attributed to the Ala, Arg,

Asn, Gly, His, Ile, Leu, Lys, Ser and Thr residues. Based on the sequences around


the chromophore-binding sites in PecB-C84-PCB and PecB(C84A)-C155-PCB from

M. laminosus, chromopeptides sequence HHRNQAAC(PCB)IRD of PecB-C84-PCB

and TKGDC(PCB)SQL of PecB(C84A)-C155-PCB were deduced. These confirm the

attachment of PCB to the correct site of PecB by CpcS and CpcT lyases.

3.5.7 Discussion

The studies described here documented the correct successful reconstitution at both

binding sites of PecB in the E. coli system. The results of support and extend the

conclusion reached by Zhao et al. (2006a, 2007 b) that the lyases CpcS and CpcT

catalyses the site- and stereoselective attachment of PCB to cysteine-β84 and 155,

respectively, in PecB.

Zhao et al. (2007b) tested whether by combined catalysis of CpcT and CpcS,

holo-β-PEC could be generated in the E. coli system. The results show that the

purified phycobiliprotein formed, however, had an absorption maximum at 602 nm,

which is red-shifted from that of of β-PEC isolated from M.laminosus (λmax = 598 nm).

By contrast, however, the fluorescence maxima were the same for the reconstituted

chromoprotein and the isolated subunit (629 nm). The PCB-C155 and PCB-C84

chromopeptide ratio of 0.7:1 was estimated of from HPLC analysis of the

chromopeptides (Fig 3-40, the sequentially reconstituted PecB-C84,C155-PCB2 ). In

β-PEC, PCB-C84 absorbs at longer wavelength than PCB-C155 and is the

predominantly fluorescing chromophore. The data thus indicate the correct

attachment of PCB to C84, but an incomplete reconstitution of PCB-C155. In

particular, doubly chromophorylated, nearly native β-PEC was again obtained only

when PCB was attached first in E. coli to C155 of PecB with CpcT, and then the

supernatant was treated with PCB and CpcS for chromophorylation at Cys-β84.

The two β subunits PecB and CpcB from M. laminosus are highly homologous (84%

similar, 68% identical)(Fig. 3-46). This similarity seems to include the interaction sites

of these subunits with both the S- and T-type lyase, because the same lyase

catalyzes the attachment of PCB to PecB and CpcB, with the same site specificily. In

this section and section 3.4, two type lyases were identified. CpcS catalyzes the

attachment of PCB to cysteine-β84 of CpcB and PecB; CpcT catalyzes the


attachment of PCB to cysteine-β155 of CpcB and PecB.

FIG. 3-46: Sequence alignment of the PecB and CpcB from M. laminosus


3.6 Structure analysis of imidazole-bilin adducts

3.6.1 Binding of PCB to CpcS

CpcS was expressed with N-terminal His-tags using the pET-30 vector to facilitate

purification and PCB binding assay. The tags did not interfere with the functions of

CpcS (Zhao et al. 2006a). When CpcS and PCB were mixed, the resulting absorption

and CD changed remarkably within 1 min (Fig.3-48), and then remained stable.

Obviously, the formation of CpcS-PCB was quick. The spectral changes indicate

strong interactions between CpcS and PCB: judged from the increase and shift of the

red absorption band, PCB is bound in a similar, extended conformation as in

phycobiliproteins. The ratio of absorption at 634 nm to that at 370 nm, and also the

inverted CD, compared to biliprotein (Fig. 3-48), are like those of PCB-ApcA2 (Fig.

3-20). The chromoprotein is only weakly fluorescent, however. The chromophore is

retained during gel filtration, where the complex moved as a monomer, indicating a

relatively stable CpcS-PCB complex (Fig. 3-47). During Ni2+- affinity chromatography,

60% of CpcS retained a PCB chromophore, the released PCB was removed in a

wash step with 1 M NaCl. If the affinity-purified complex is denatured by acidic urea,

the absorption maximum (690 nm, Fig.3-48) was that of free PCB carrying a

3-ethylidene group (Storf et al. 2001).This indicated that most of PCB was bound

non-covalently with CpcS, or is bound so weakly that it is lost in the acidic solution.

The complex was therefore analyzed by proteolysis with trypsin under neutral

conditions, and subsequent HPLC and mass spectrometry (Fig.3-51)

300 400 500 600 700 8000 .00

0 .05

0 .10

0 .15

0 .20

0 .25

0 .30

Abs

orpt

ion

[a.u

.]

W avelength [nm ]

C pcS -P C B 0 pH 7 .5 1% fo rm ic ac id 2% fo rm ic ac id 5% fo rm ic ac id 10% fo rm ic ac id

FIG. 3-47: Absorption of CpcS-PCB in different concentration formic acid in KPB (500 mM, pH 7.5) containing NaCl (100 mM).


300 400 500 600 700 800

0

10

20

CD

[mde

g]

Wavelength [nm]300 400 500 600 700 800

0.2

0.4

Abso

rptio

n

Wavelength [nm]

A B

FIG. 3-48: Binding of PCB to CpcS. Absorption (A) and CD (B) changes on formation of CpcS-PCB. Spectra of PCB (5 μM) in KPB (500 mM, pH 7.5) containing NaCl (100 mM) (- - - -), plus CpcS (15 μM) in the same buffer recorded 1 min after mixing (⎯⎯), this spectrum remained unchanged for 2 hr, and absorption of the same solution after addition of acidic urea (8 M, pH 2) (• • • •).

3.6.2 Imidazole binding PCB

When CpcS, PCB and imidazole were mixed, the resulting absorption changed

remarkably within 120 min (Fig.3-49), and then remained stable. The spectral

changes indicate strong interactions between CpcS-PCB and imidazole, judged from

the decrease and blue shift of the vis absorption band (from 634 nm to 605 nm). The

formed complex was named CpcS-PCB-imidazole; it is only weakly fluorescent. The

affinity-purified complex is denatured by acidic urea, the absorption maximum (660

nm, not shown) was that of PCB that is covalently attached via C-31 to a thiol,

including cystein in a protein. The CD spectrum of the complex is like that of

PCB-CpcS (Fig. 3-50). CpeS-PCB-imidazole was digested with trypsin and then

analyzed by HPLC with diode-array detection (Fig. 3-51). The colored bands 1 and 2

did only contain traces of several peptides that could not be identified with certainty;

the major component is a IPCB-imidazole adduct according to MS (see 3.6.5). This

indicated that most of PCB was bound covalently to the imidazole, and that

imidazole-PCB was bound non-covalently to CpcS.


300 400 500 600 700 8000.0

0.5

1.0

1.5

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

1 min 30 min 60 min 90 min 120min 150min 180min 210min 240min

FIG. 3-49: Binding of PCB to imidazole. Absorption changes on formation of CpcS-PCB-imidazole after mixing PCB (5 μM), CpcS (15 μM) plus imidazole(500 mM) in KPB (500 mM, pH 7.5) containing NaCl (100 mM). Sample was maintained at 35 °C throughout.

300 400 500 600 700 800-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

CD [a

.u.]

Wavelength [nm]

200 220 240 260 280 300-4.0

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

CD [a

.u.]

Wavelength [nm]

FIG. 3-50: CD spectra of CpcS-PCB-imidazole Vis-CD (top) and far-UV CD spectra (bottom) of complex CpcS-PCB-imidazole was purified by Ni2+ affinity chromatography and dialyzed against KPB (20 mM, pH 7.2)


Since imidazole is absent in CpcS untreated with PCB under the same conditions (not

shown), this indicated that residual imidazole was involved and bound with high

affinity during the reaction with PCB. To verify this possibility, we repeated the

experiment of PCB binding in the presence of increasing concentrations of imidazole;

the amounts of bands 1 and 2 containing the IPCB-imidazole adduct increased with

imidazole concentration, at the expense of free PCB (Fig. 3-51). Band 1 adduct was

named iPCB-imidazole 21 according to its tr and structure (3.6.6), band 2 adduct was

named iPCB-imidazole 23. To verify the difference of iPCB-imidazole 21 and

iPCB-imidazole 23, we repeated the experiment of PCB binding imidazole at different

temperature. The results showed increasing amounts of iPCB-imidazole 23 with

increasing temperatures, at the expense of free PCB and iPCB-imidazole 21 (Fig.

3-52). Bases on the maximum absorption of iPCB-imidazole 21 (λmax=633 nm) and

iPCB-imidazole 23 (λmax=648 nm), iPCB-imidazole 23 may be an iPCB-imidazole 21

oxidation product.

300 400 500 600 700 800

0.05

0.10

0.15

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

B

10 20 30 40 50

0.20.0

0.2

0.0

0.2

0.0

0.2


Abso

rptio

n [a

.u.]

A

0 mM

100mM

Imidazole 500 mM

1 23

200mM

FIG. 3-51: Dependence of colored products formed during reaction of CpcS, PCB and imidazole on imidazole concentration. HPLC of colored products from tryptic digestion of CpcS-PCB-imidazole. CpcS (30 μM), PCB (10 μM) and various concentrations (0, 100, 200, 500 mM) of imidazole were incubated in KPB (500 mM, pH 7.5) containing NaCl (100 mM) at 35 °C for 3 hr. The resulting complexes were purified by Ni2+ affinity chromatography and dialyzed against KPB (20 mM, pH 7.2). The purified complexes (10 μM) were then digested with trypsin (40 μM) at 37 °C for 3 hr, and analyzed by HPLC (Zhao et al. 2007). A: HPLC traces with absorption set to 650 nm, B: absorption spectra of peak 1 (⎯⎯), peak 2 (- - - -) and peak 3 (• • • •) recorded in line by diode array detection (Tidas, J&M, Aalen).


300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

0.25

Abso

rptio

n [a

.u.]

Wavelength [nm]


B

10 20 30 40 500.00

0.05

0.10

0.150.00

0.05

0.10

0.150.00

0.05

0.10

0.15A

bsor

ptio

n [a

.u.]


20℃

37℃

50℃1 23A

FIG. 3-52: Temperature dependence of colored proucts formed during reaction of CpcS, PCB and imidazole. HPLC of colored products from tryptic digestion of CpcS-PCB-imidazole. CpcS (30 μM), PCB (10 μM) and imidazole (500 mM) were incubated in KPB (500 mM, pH 7.5) containing NaCl (100 mM) at various temperature for 1 hr. Sample preparing methods see Fig. 3-51. A: HPLC traces with absorption set to 650 nm, B: absorption spectra of peak 1 (⎯⎯), peak 2 (- - - -) and peak 3 (• • • •) recorded in line by diode array detection (Tidas, J&M, Aalen).

3.6.3 Chromophore transfer from CpcS-PCB to CpcB(C155I)

The weak fluorescence of CpcS-PCB opened a way to test the chromophore transfer

from CpcS-PCB to the final acceptor, CpcB(C155I), because the chromophores

become highly fluorescent after correct binding to the native protein. CpcB(C155I)

was used as acceptor; the second binding site at cysteine155 was removed by

site-directed mutagenesis to avoid complications from unspecific binding (Fig. 3-53).

As evidenced by the increase in fluorescence, and by the blue-shifted absorption, the

bound PCB was transferred from CpcS-PCB to cysteine-84 of the apoproteins

yielding PCB-CpcB(C155I). The spectral features of the final product were identical to

those published for the respective chromoprotein (Zhao et al. 2006a). CpcS-PCB

therefore seems to be an intermediate in the attachment reaction of PCB to

CpcB(C155I) catalyzed by CpcS.


300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

20

40

60

80

100

Fluo

resc

ence

[a.u

.]

FIG. 3-53: Chromophore transfer from CpcS-PCB to CpcB(C155I). Absorption (⎯⎯) and fluorescence (- - - -) after mixing of CpcS-PCB (2 μM) with CpcB(C155I) (10 μM). The first spectrum was recorded 1 min after mixing, the subsequent ones every 20 min up to 100 min. The sample was maintained at 35 °C throughout; arrows indicate the spectral changes during the reaction.

3.6.4 Chromophore transfer from imidazole-bilin adducts to

CpcB(C155I), CpcS or mercaptoethanol

3.6.4.1 Chromophore transfer from CpcS-PCB-imidazole to CpcB(C155I)

During the transfer reaction, the absorption maximum of CpcS-PCB-imidazole shifted

significantly from 605 to 618 nm (Fig. 3-54), indicating that the attachment reaction

was nearly complete in 3hr. Incubation of CpcS-PCB-imidazole with CpcB(C155I)

resulted in the induction of a red fluorescence of the emission maximum at 645 nm.

The spectral features of product are similar that of PCB-CpcB(C155I) (Zhao et al.

2006a), indicating that PCB was transferred from CpcS-PCB-imidazole to Cys-84 of

CpcB(C155I), and formed PCB-CpcB(C155I).


0

100

200

300

400

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Wavelength [nm]

Abs

orpt

ion

[a.u

.]

1 min 1 hr 3 hr 5 hr

FIG. 3-54: Chromophore transfer from CpcS-PCB-imidazole to CpcB(C155I). Absorption (left) and fluorescence (right) after mixing of CpcS-PCB-imidazole (2 μM) with CpcB(C155I) (10 μM). The first absorption spectrum was recorded 1 min after mixing, the subsequent ones every 1~2hr up to 5hr, the fluorescence spectrum was recorded 5hr after mixing. Sample was maintained at 35 °C throughout.

3.6.4.2 Chromophore transfer from iPCB-imidazole to CpcS

During the transfer reaction, the absorption maximum of iPCB-imidazole shifted

significantly from 600 to 634 nm (Fig. 3-55), indicating that the attachment reaction

was nearly complete in 2hr. The spectral features of product are similar that of

PCB-CpcS (see above), indicating that PCB was transferred from iPCB-imidazole to

CpcS, and formed PCB-CpcS. Although PCB is bound non-covalently to CpcS, the

equilibriumis shifted from the covalently-bound adduct, iPCB-imidazole.

300 400 500 600 700 800-0.02

0.00

0.02

0.04

0.06

0.08

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

1 min 60 min 120min

300 400 500 600 700 800-0.02

0.00

0.02

0.04

0.06

0.08

0.10 1 min 60 min 120min

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

A B

FIG. 3-55: Chromophore transfer from iPCB-imidazole to CpcS. Absorption after mixing of iPCB-imidazole 21(A) or iPCB-imidazole 23(B) (2 μM) with CpcS (10 μM). The first spectrum was recorded 1 min after mixing, the subsequent ones every 1hr up to 2hr. Sample was maintained at 35 °C throughout.


3.6.4.3 Chromophore transfer from iPCB-imidazole to CpcB(C155I)

Last, iPCB-imidazole, was incubated with both CpcS and CpcB(C155I). The ensuing

spectral chances are shown in Fig. 3-56. The absorption maximum of iPCB-imidazole

shifted significantly from 600 to 618 nm, indicating that the attachment reaction was

nearly complete in 2hr. Incubation of iPCB-imidazole, CpcS and CpcB(C155I) also

resulted in the induction of a red fluorescence of the emission maximum at 645 nm.

The spectral features of final product are similar that of PCB-CpcB(C155I) (Zhao et al.

2006a), indicating that PCB was transferred from iPCB-imidazole to CpcB(C155I),

and formed PCB-CpcB(C155I).

300 400 500 600 700 8000.00

0.04

0.08

0.12

0.16

0.20

Wavelength [nm]

Abs

orpt

ion

[a.u

.]

0

100

200

300

400

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 800

0.00

0.05

0.10

0.15

0.20

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

100

200

300

400

500

Fluo

resc

ence

a.u

.]

A B

FIG. 3-56: Chromophore transfer from iPCB-imidazole to CpcB(C155I). Absorption (left) and fluorescence (right) after mixing of iPCB-imidazole 21(A) or iPCB-imidazole 23(B) (2 μM), CpcS (10 μM) with CpcB(C155I) (10 μM). The Absorption spectrum was recorded 1 min and 2hr after mixing, the fluorescence spectrum was recorded 2hr after mixing. Sample was maintained at 35 °C throughout.

3.6.4.4 Chromophore transfer from iPCB-imidazole to mercaptoethanol

Imidazolides, viz. amides resulting from the reaction of imidazole with carboxylic acids,

are generally very labile; we therefore also tested the reactivity of iPCB-imidazole with

mercaptoethanol. When iPCB-imidazole and mercaptoethanol were mixed, the

maximum absorption of the mixture did not change within 120 min (Fig. 3-57 A, B),

indicating that the chromophore was not cleaved from iPCB-imidazole complex, and

transferred to mercaptoethanol. To verify this stability, we reconcentrated and HPLC

tested the reaction product (Fig. 3-57 a, b). The figures showed that the retention time

and the maximum absorption of reaction product is same of that of the initial

iPCB-imidazole complex, and not reaction took place to mercaptoethanol. In view of

the lability of imidazolides (Schaltiel, 1967), this indicates a different binding mode of

imidazole to PCB


10 20 30 400.00

0.05

0.10

0.15

Abs

orpt

ion

[a.u

.]


300 400 500 600 700 8000.00

0.04

0.08

0.12

0.16

0.20

no ME 1min 1hr 2hr

Abs

orpt

ion

[a.u

]

Wavelength [nm]

300 400 500 600 700 8000.00

0.05

0.10

0.15

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

no ME 1min 1hr 2hr

10 20 30 400.00

0.05

0.10

0.15

Abs

orpt

ion

[a.u

]


300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

300 400 500 600 700 8000.00

0.05

0.10

0.15

Abs

orpt

ion

Wavelength(nm)

aA

B b

FIG. 3-57: Absence of chromophore transfer from iPCB-imidazole to mercaptoethanol and HPLC analysis. Absorption after mixing of iPCB-imidazole 21(A) or iPCB-imidazole 23(B) (2 μM) with mercaptoethanol (6 mM).The first spectrum was recorded 1 min after mixing, the subsequent ones every 1hr up to 2hr. Sample was maintained at 35 °C throughout.The resulting product (iPCB-imidazole 21(a) and iPCB-imidazole 23(b)) were concentrated with Sep-Pak cartridges and analyzed by HPLC (Zhao et al. 2007a)

3.6.5 HPLC–ESI-MS analysis of imidazole-bilin adducts

CpcS-PCB-imidazole and CpcS-PCB were digested with trypsin and then analyzed by

HPLC with diode-array detection (Fig.3-51). Most of the pigments in the digest of the

purified CpcS-PCB (>95%, band 3 in Fig.3-51) was free PCB, as identified by its

retention time, its absorption at 690 nm and mass spectroscopy (table 3-21). Most of

the colored products in the digest of the purified CpcS-PCB-imidazole (>60%, band1

and band 2 in Fig.3-51) were iPCB-imidazole adducts. The faster moving bands 1 and

2 did contain traces of several peptides that could not be identified with certainty.


Table 3-21: Molecular masses (m/z) of colored bands in tryptic digest of CpcS-PCB-imidazole or CpcS-PCB

There is excellent agreement between the measured iPCB-imidazole 21 Mr values

and the Mr values calculated for a iPCB-imidazole addition product (Table 3-21),

which proves PCB was bound covalently with the imidazole. It disproves at the same

time the formation of an imidazolide (Mrcal= 637m/z). The major peak at 655.321 m/z

represents the singly charged PCB adduct PCB-imidazole. The largest peak in band 2

had a mass of 655.311 m/z matching exactly that of band 1, this is therefore an

isomeric PCB-imidazole addition product. Band 2 shows a second peak of 50%

intensity that is smaller by 2 mass units (Table 3-21), possibly it relates to an oxidation

product of the former. Addition products of PCB are known to oxidize readily to

mesobiliverdins (Fairchild et al., 1992), the ease of this may depend on the

stereochemistry. Peak 653.31 m/z was analyzed by MSMS, indicating that a

chromophore of m/z = 585.31 is formed that is 2 mass units smaller than PCB, i.e. the

mass of mesobiliverdin.

3.6.6 NMR analysis of imidazole-bilin adducts

For NMR spectroscopy, the iPCB-imidazole 21 and iPCB-imidazole 23 were purified

by HPLC again. RP-HPLC separation of PCB adduct was achieved on a C18 column.

The 1H-NMR spectrum of iPCB-imidazole is shown in Fig. 3-58. All assignments refer

to the A-ring linkage numbering scheme (see Fig. 3-59) and summarized in Table

3-22.

Mass (m/z) Peak

in Fig. 3-51

Species

Calc. Expt.(intensity)

1 iPCB-imidazole [PCB+imidazole+H]+ 655.324 655.321 (100%)

iPCB-imidazole [PCB+imidazole +H]+ 655.324 655.311(100%)

2 Oxidation product

chromophore

[PCB-imidazole-2H +H]+

[PCB-2H+H]+

653.309

585.290

653.330(50%)

585.306(10%)

3 PCB [PCB+H]+ 587.290 587.301(100%)




A

B

FIG. 3-58: 1-D 1H-NMR spectra of iPCB-imidazole 21 (A) and iPCB-imidazole 23 (B) in 10 mM TFA/D2O

The NMR-spectra (Fig. 3-58A) result from a combination of the chromophore and

imidazole signals (Table 3-22). The downfield region of the spectrum shows three

vinylic10-H, 15-H and 5-H resonances at 7.00, 6.23 and 5.43 ppm, respectively, these

peaks are like those of the A-ring-linked α-1 PCB chromophore (Bishop et al., 1986).

The 31-H quartet at 4.06 ppm is downfield shifted due to its proximity to the imidazole

nitrogen, confirming the attachment at this site. The 31-H is coupled to the 32-CH3

group (yielding a doublet at 1.61 ppm). Similarly the 2- CH3 signal (1.15 ppm) is

coupled to the 2-H proton (3.30 ppm).Remarkably, however, neither the 31-H nor the

2-H signals couple to a second set of protons, indicating that there is no 3-H, in

agreement, there is no 3-H signal in spectrum.



of the spectra of bile pigments. The three aromatic methyls at C-7, -13, and -17

singlets resonate at 2.11, 2.10 and 1.97 ppm (singlets). The ethyl group at C-18 is

seen as a multiplet at 2.32 ppm (18- CH2CH3) and a triplet at 0.98 ppm (18-CH2CH3).

The multiplet resonances from methylene group of the propionic acid side chains at

C-8 and C-12 were found at 2.89 and 2.37 ppm. The three vinylic 2-H, 4-H and 5-H of

imidazole resonate at 7.45, 7.28 and 7.18 ppm, thus confirming, the binding via one of

N-atoms. Based on this analysis of the NMR spectrum of iPCB-imidazole 21, we

propose the following structure of iPCB-imidazole 21(Fig. 3-59).


Table 3-22: 1H NMR(600-MHz) assignments for iPCB-imidazole 21 and iPCB-imidazole 23 (10 mM TFA/D2O).

NH

NH HN

COOHCOOH

HN

OOH

A

BC

D

1 2

34

5

6

1819

17

16

15

14

31

7

89

101112

13

N N

FIG. 3-59: Structure of iPCB-imidazole 21 (bound-2,22-DBV)

Chemical shift (ppm) iPCB-imidazole 21 iPCB-imidazole 23

(major(minor))


Number of H’s

Assignment

7.45 7.45 br 1 Imidazole 2-H 7.28 7.31(7.27) br 1 Imidazole 4-H 7.18 7.20 br 1 Imidazole 5-H 7.00 6.97 s 1 10-H 6.28 6.31(6.22) s 1 15-H 5.43 5.96(5.71) s 1 5-H

4.06 4.06 q 1 3’-H 3.30 3.30 m 1 2-H 2.89 2.89(2.83) m 4 8,12-CH2CH2COOH 2.37 2.36(2.34) m 4 8,12-CH2CH2COOH2.32 2.26(2.19) m 2 18- CH2CH3 2.11 2.12(2.11) s 2.10 2.09(2.07) s 1.97 1.98(1.96) s

9 7,13,17-CH3

/ 1.80 s 3 2- CH3 1.61 1.67 d 3 3’- CH3 1.15 1.12 d 3 2-CH3 0.98 1.02(0.97) t 3 18- CH2CH3


Parallel analysis of the NMR spectra of iPCB-imidazole 21 and iPCB-imidazole 23

(Table 3-22) shows, like the MS-spectrum, that iPCB-imidazole 23 is a 100:60 mixture

of two compounds. The major one resembles closely that of iPCB-imidazole 21. There

is in particular again no 3-H visible; however, most bands are somewhat thifted

compared to iPCB-imidazole 21. The resonances attributed to the aromatic methyls of

C-7, -13, and -17, the three vinylic ones of C-5, -10, and -15, the ethyl group of C-18,

and the propionic acid methylenes at C-8 and -12 are salient features of the spectra of

both pigments. For the minor compound, we found the 2- CH3 singlet at 1.80 ppm and

the 31-H quartet at 4.06 ppm in the NMR spectrum of iPCB-imidazole 23, indicating a

double band between the C2 and C3. This indicates that the larger part of the mixture

is a iPCB-imidazole adduct that is isomeric to iPCB-imidazole 21, and the minor

component is the oxidation product, most likely mesobiliverdin (Fig. 3-60). It remains

unclear whether the latter is formed during the addition reaction, or only later during

workup and chromatography.

NH

NH HN

COOHCOOH

HN

OOH

A

BC

D

1 2

34

5

6

1819

17

16

15

14

31

7

89

101112

13

N N NH

N HN

COOHCOOH

HN

OOA

BC

D

1 2

34

5

6

1819

17

16

15

14

31

7

89

1011

12

13

N N

+

FIG. 3-60: The structure of iPCB-imidazole 23 complex

3.6.7 Discussion

Binding of chromophores to lyases has been observed before with both subunits of

the E/F-type lyases, but there binding was relatively weak, and most of the

chromophore was lost during Ni2+-affinity chromatography (Zhao et al. 2006). CpcS

binds PCB strongly enough that ~60% are retained in this step during purification, and

even a considerable amount (~5%) is retained during SDS-PAGE. However, also in

this case most of the chromophore is lost during tryptic digestion, indicating only a

weak, probably non-covalent bond compared to the thioether bond present in

phycobiliproteins.


The iPCB-imidazole adducts, iPCB-imidazole 21 and iPCB-imidazole 23, were found

serendipitously resulting from the presence of residual imidazole in the Ni2+-affinity

purified CpcS. The structural significance of the iPCB-chromophore is discussed in

the next chapter. The adduct formation indicates that CpcS can promote covalent

binding of PCB to imidazole, and thereby possibly to histidine. In the phytochromes,

which bind the tetrapyrrole chromophore autocatalytically, a histidine is present near

the ‘classical’ binding site around aa 250, which is even conserved, and important for

binding, in the new type of phytochromes where the binding cysteine is near the

N-terminus (Lamparter et al. 2004). The CpcS-catalyzed binding of PCB to imidazole

may relate to these observations, such that the imidazole of a histidine residue binds

first to the chromophore, and this in turn promotes binding of the chromophore to

another amino acid. Judged from the absorption and NMR spectra of iPCB-imidazole

21 and iPCB-imidazole 23, the chromophore is bound in both products via the

3-ethylidene group. It therefore appears that the ethylidene group present in free PCB

can add other amino acids than cysteine, but that the resulting C-N bonds are less

stable than the thioether bond resulting from addition of cysteine. There are several

histidines in CpcS from Anabaena PCC7120, but only one near the N-terminus is

conserved; there is no conserved histidine among CpcB and PecB. It is conceivable

that the conserved histidine in CpcS can add to the ethylidene double-bond of PCB.

Chromophorylation by CpcS might then involve a histidine-bound intermediate; this

could be a model for the reaction catalysed by CpcS.

PCB binding to CpcS is fast compared to the lyase-catalyzed chromophorylation of

the apoprotein. Kinetic constants for the reactions of all three types of lyases were in

the range of 20 min or more (Fairchild and Glazer 1994; Zhao et al. 2006a,b), and

even the binding in phytochrome (Borucki et al. 2003) is much slower than the

reaction seen with CpcS, which is complete after less than one minute. This indicates

that the rate limiting step in the catalytic reaction of CpcS is the transfer to the

accepting apoprotein, in this case CpcB. This time (100 min) is very similar to the time

constant of the full reaction of CpcS catalyzed chromophore attachment to CpcB.

Protein-protein interaction has also been found rate limiting in the EF-Type lyases.

Here, the rate of chromophore addition by the E-subunit was much enhanced when it

was pre-incubated with the acceptor protein, PecA (Böhm et al. 2007). However, even

in this case, the reaction is more than an order of magnitude slower than


chromophore binding by CpcS.

The chromophore transfer from CpcS-PCB to the acceptor apoprotein is the first solid

evidence that chromophore binding to CpcS is part of the enzymatic activity. Evidence

for such a transfer has also been obtained for the EF-type lyases (Zhao et al. 2006),

but the reaction is much more clear-cut with CpcS. Böhm et al. (2007) have recently

proposed a chaperone-like action for the E-subunit of the EF-type lyases. Covalent

binding is not necessarily contradicting such a mechanism. If the lyase function

involves the proper conformational control of the chromophore (Zhao et al. 2004), this

may well be assisted by covalent binding to the lyase, as long as this is reversible. A

native-like conformation of the chromophore bound to CpcS, is evidenced in this work

by the absorption increase in the visible region, and by the CD of CpcS-PCB. A

relatively loose binding is finally indicated by the weak fluorescence, and by transfer

of the chromophore also to imidazole and mercaptoethanol (see Chapter 3.7)

In summary, PCB binds fast to CpcS, and the chromophore of the resulting

CpcS-PCB complex is transferred much more slowly to the Cys84 of the acceptor

apoprotein, CpcB. The addition of imidazole to PCB, catalyzed by CpcS, yields two

isomeric iPCB-imidazole adducts that are non-covalently bound to CpcS. CpcS

catalyses chromophore transfer of the two complexes, with concomitant

re-isomerization to PCB, to Cys84 of the acceptor apoprotein, CpcB. The

chromophore can also be transferred from the two complexes to CpcS, yielding

CpcS-PCB. Based on these experimental results, we propose the following reaction

schemes among the CpcS, PCB, CpcB(C155I) and imidazole (Fig. 3-61) .

CpcS-iPCB-imidazole

PCB CpcS-PCB PCB-CpcB(C155I)

iPCB-imidazole

fast

Trypsin

+CpcS

+imidazole

+CpcB(C155I)

+CpcS+CpcB(C155I)+CpcS

imidazole

CpcS

FIG. 3-61: Reaction schemes of CpcS, PCB, CpcB(C155I) and imidazole.


3.7 Structure analysis of mercaptoethanol-bilin adducts

3.7.1 Mercaptoethanol binding PCB catalyzed by CpcS

When CpcS, PCB and mercaptoethanol were mixed, the resulting absorption and

fluorescence spectra changed remarkably within 180 min (Fig.3-62), and then

remained stable. The spectral changes indicate strong interactions between CpcS,

PCB and mercaptoethaol, judged from the decrease and blue shifted absorption band

(from 634 nm to 597 nm) and the increase of fluorescence (λmax = 630 nm). The

formed complex was named CpcS-PCB-mercaptoethanol. If the affinity-purified

complex is denatured by acidic urea, the absorption maximum (660 nm, not shown)

was like that of PCB attached via C-31 to a cysteine in a chromoprotein or

chromopeptide. The CD spectrum of the complex (594 nm (-)/373 nm (+)) is like that

of PCB-CpcS (Fig. 3-63). CpeS-PCB-mercaptoethanol was purified by Ni2+ affinity

chromatography and dialyzed against KPB (20 mM, pH 7.2). The purified complex

was digested with trypsin and then analyzed by HPLC with diode-array detection (Fig.

3-64). The colored band 1 (tr=29.1 min) had an absorption maximum at 648 nm and

band 2 (tr=30.3 min) had an absorption maximum at 633 nm that are typical for

PCB-containing products. The products contained in colored band 1 and 2 adduct

were named iPCB-ME 29 and iPCB-ME 31, respectively, according its retention time

and structure (see section 3.7.4). The colored band 3 (tr= 34.1 min) corresponds to

free PCB according to its retention time and the absorption maximum at 684 nm.

iPCB-ME 29 and iPCB-ME 31 did contain traces of several peptides that could not be

identified by mass spectrometry with certainty. The digestion results indicated that

most of PCB was not bound covalently to CpcS (or only very weakly), but that rather

some other modification had happened. As will be shown in section 3.7.4, both

products an indeed addition products of PCB with ME, with the same modified iPCB

chromophore as in the imidazole adducts described in the previous chapter.


300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

1 min 30 min 60 min 120 min 180 min

0

100

200

300

Fluo

resc

ence

[a.u

.]

FIG. 3-62: Binding of PCB to mercaptoethanol. Absorption (left) and fluorescence (right) after mixing of CpcS (30 μM), PCB (10 μM) and mercaptoethanol (6 mM). The first spectrum was recorded 1 min after mixing, the subsequent ones every 30-60 min up to 180 min. Sample was maintained at 35 °C throughout.

200 220 240 260 280 300-4

-2

0

2

CD [a.u.]

Wavelength [nm]

300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

Abs

orption [a.u.]

Wacelength [nm]

300 400 500 600 700 800-15

-10

-5

0

5

10

15

20

CD [a.u.]

Wavelength [nm]

FIG. 3-63: CD spectra of CpcS-PCB-mercaptoethanol Absorption (top), Vis- CD (middle) and far-UV CD spectra (bottom) of complex CpcS-PCB-mercaptoethanol was purified by Ni2+ affinity chromatography and dialyzed against KPB (20 mM, pH 7.2)


300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Abs

orpt

ion

[a.u

.]

Wavelength [nm]


10 20 30 40 500.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40


Abs

orpt

ion@

650n

m [a

.u.] 1 2

3

FIG. 3-64: HPLC of colored products from tryptic digestion of CpcS-PCB-mercaptoethanol reaction mixture. CpcS (30 μM), PCB (10 μM) and ME (60 mM) were incubated in KPB (500 mM, pH 7.5) containing NaCl (100 mM) for 3hr. The resulting complexes were purified by Ni2+ affinity chromatography and dialyzed against KPB (20 mM, pH 7.2). The purified complexes (10 μM) were then digested with trypsin (40 μM) at 37 °C for 3 hr, and analyzed by HPLC (Zhao et al. 2007).

3.7.2 Spontaneous binding of mercaptoethanol to PCB

To test the possibility of a chemical reaction of PCB with ME, both were incubated in

the absence of CpcS. When PCB and mercaptoethanol were mixed, the absorption

decreased and blue shifted from 625 nm to 595 nm within 120 min (Fig.3-65), and

then remained stable. The fluorescence (λmax = 697 nm) did not change and remained

very weak. The formed complex was named PCB-mercaptoethanol.

PCB-mercaptoethanol was concentrated by Sep-Pak cartridges and analyzed by

HPLC (Zhao et al. 2007) (Fig. 3-66). It yielded four distinct colored bands, one

seemed pure, the other three were mixtures judged from the band-shape that could

not be further separated. The colored band A (tr=27.9 min) had an absorption

maximum at 564 nm and band B (tr=28.9 min) had an absorption maximum at 584 nm

both are in the range of PVB-containing products. They were named PVB-ME 27 and

PVB-ME 28, respectively, according their retention times and structures (see section

3.7.4). The colored bands C and D correspond to iPCB-ME 29 and iPCB-ME 31

obtained in the reaction with ME, PCB and CpcS (Fig. 3-62) according to their


retention times and the absorption maximum. This indicated that PCB was bound

covalently, in a spontaneous reaction, with the mercaptoethanol and formed 4 kinds of

colored products PVB-ME 27, PVB-ME 28, iPCB-ME 29, iPCB-ME 31, but that in the

presence of CpcS mainly the two iPCB-adducts were formed. The structures of all

four complexes were analyzed by mass and NMR spectroscopy.

300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orpt

ion

[a.u

.]

Wavelength [nm]


0

2

4

6

8

10

Fluo

resc

ence

[a.u

.]

FIG. 3-65: Binding of PCB to mercaptoethanol. Absorption (left) and fluorescence (right) after mixing of PCB (10μM) and mercaptoethanol (6 mM). The first spectrum was recorded 1 min after mixing, the subsequent ones every 30-60 min up to 180 min. Sample was maintained at 35 °C throughout. The arrow indicates the spectra changes during the reaction.

10 20 30 40 500.00.20.40.6

0.00.20.40.60.0

0.2

0.40.00.20.40.6

D

Abs

orpt

ion@

650n

m


A

C

B

Abs

orpt

ion@

600n

m


300 400 500 600 700 8000.0

0.2

0.4

0.6A

bsor

ptio

n [a

.u.]

Wavelength [nm]

A B C D

FIG. 3-66: HPLC and in situ absorption of PVB-ME 27(A) , PVB-ME 28 (B), iPCB-ME 29 (C) and iPCB-ME 31 (D) obtained from the reaction PCB with mercaptoethanol.

3.7.3 Structure analysis of mercaptoethanol-bilin adducts

All four products isolated from the reaction of PCB and ME had identical masses

(Table 3-23) that are the sum of both components. They are therefore isomeric

addition products.

Table 3-23: Molecular masses (m/z) of isolated mercaptoethanol-bilins from Fig. 3-66

In order to determine the chromophore structure and mode of linkage to

mercaptoethanol, the 600-MHz 1H NMR spectra of the ME-bilin products (PVB-ME 27,

PVB-ME 28, iPCB-ME 29 and iPCB-ME 31) in 10 mM TFA/D2O, were compared (Fig.

3 -67). All assignments refer to the A-ring linkage numbering scheme (see Fig. 3-69)

and are summarized in tables 3-24 and 3-25.

Mass (m/z) Peak Species

Calc. Expt.

A PVB-mercaptoethanol [PCB+ME+H]+ 665.431 665.245

B PVB-mercaptoethanol [PCB+ME +H]+ 665.431 665.226

C iPCB-mercaptoethanol [PCB+ME +H]+ 665.431 665.236

D iPCB-mercaptoethanol [PCB+ME +H]+ 665.431 665.264


8 7 6 5 4 3 2 1 0

8 7 6 5 4 3 2 1 0

chemical shift [ppm]

8 7 6 5 4 3 2 1 0

8 7 6 5 4 3 2 1 0

A

B

C

D

FIG. 3-67: 1-D 1H-NMR spectra ( up) and 31-H spectra (bottom) of PVB-ME 27(A) , PVB-ME 28 (B) , PCB-ME 29 (C) and PCB-ME 31 (D) in 10 mM TFA/D2O

Parallel analysis of the NMR spectra of PVB-ME 27 and PVB-ME 28 reveals for both

well defined resonances of the chromophore (Table 3-24), the assignment of all

relevant resonances of a PVB group can be made. Analyzing the downfield region of

the PVB-ME 27 (PVB-ME 28) spectrum, one observes only two vinylic 10-H and 15-H

resonances at 7.08 (7.11) and 6.29 (6.28) ppm, respectively. These peaks

correspond well to the 10-H and 15-H singlets previously observed in the A-ring-linked

peptide α-1 PVB from Anabaena PCC7120 (see Charpter 3.1). Noticeably absent is a

third downfield vinylic resonance corresponding to 5-H. PVB lacks the methine bridge

between rings A and B and, therefore, shows no such resonance, the lack of the

respective signal is taken as proof of saturation between the A and B rings. Because

the mass spectral data identified both PVB-adducts as addition products, another

non-conjugated double bond must exist elsewhere on the PVB-tetrapyrrole moiety.

The only alternative position for this unsaturation is between C-2 and C-3, which is


corroborated by the 1H NMR spectrum. There are four aromatic methyl singlets at

1.76(1.79), 1.99(2.01), 2.08(2.08) and 2.12(2.12) ppm for C-2, -7, -13, and -17,

thereby proving the presence of a double bond between C-2 and C-3, as in PVB.

The doublet at 1.47(1.46) ppm is assigned to 31-CH3 and a quartet at 4.06(4.06) ppm

is assigned 31-H. The resonances from the propionyl side chains are found at

2.89(2.90) and 2.35(2.36) ppm. The ethyl group is seen as two multiplet at 2.31(2.31)

ppm (18- CH2CH3) and 1.01(1.00) ppm (18-CH2CH3). The resonance of 4-H was

hidden under the resonance of the solvent, and coupling with the two non-equivalent

5-H protons at 3.49(3.42) and 2.82(2.73) ppm is obvious. The resonance of α-CH2

were found at 3.60(3.61) and β-CH2 at 2.59(2.56) from mercaptoethanol. There are

major difference (0.07-0.08 ppm) of the two non-equivalent 5-H protons, and smaller

ones (< 0.03 ppm) of the other protons between PVB-ME 27 and PVB-ME 28. We

propose the following structure of PVB-ME 27 and PVB-ME 28 (Fig. 3-69). They are

isomeric PVB-ME addition products, with different (relative) stereochemistries at C-31

and C-4.

Table 3-24: 1H NMR (600-MHz) assignments for PVB-ME 27 and PVB-ME 28 (10 mM TFA/D2O).

Chemical shift (ppm) PVB-ME 27 PVB-ME 28


Number of H’s

Assignment

7.08 7.11 s 1 10-H 6.29 6.28 s 1 15-H

/ / / 4-H 4.06 4.06 q 6.98(6.95) 1 3’-H 3.60 3.61 m 2 ME α-CH2 3.49 3.42 m 1 5-H 2.89 2.90 m 4 8,12-CH2CH2COOH 2.82 2.73 m 2 5-H 2.59 2.56 m 1 ME β-CH2 2.35 2.36 m 4 8,12-CH2CH2COOH 2.31 2.31 m 2 18- CH2CH3 2.12 2.12 s 3 17-CH3 2.08 2.08 s 3 13-CH3 1.99 2.01 s 3 7-CH3 1.76 1.79 s 3 2-CH3 1.47 1.46 d 6.92(7.28) 3 3’- CH3 1.01 1.00 t 3 18- CH2CH3


NH

N HN

COOHCOOH

HN

OOA

BC

D

1 2

34

5

6

1819

17

16

15

14

31

7

89

1011

12

13

*

* S

CH2

CH2

OH

FIG. 3-69: Structure of PVB-ME 27 and PVB-ME 28 (bound PVB), * indicate asymmetric C-atoms

Parallel analysis of the NMR spectra of iPCB-ME 29 and iPCB-ME 31 gives again

very similar spectra for the two products (Table 3-25), which are assigned to isomers

of adducts containing a novel a 2,22-DBV-ME(=iPCB) chromophore. The downfield

region of the spectrum shows three vinylic 10-H, 15-H and 5-H resonances at

6.94(6.94), 6.30(6.30) and 5.89(5.73) ppm, respectively. The 31-H quartet at 4.06

(4.06) ppm is downfield shifted due to its proximity to the thiol, comfirming the

attachment at this site. The 31-H is coupled only to the 31-CH3 group (doublet at

1.38(1.25) ppm). Similarly the 2- CH3 shows as a quarlet (1.21(1.17) ppm) that is only

coupled to the 2-H proton (3.42(3.44) ppm). Remarkably, neither the 31-H nor the 2-H

singals therefore couple to a second set of protons, indication that there must be a

double bond at C-3.

All other signals attributed to the chromophore are similar to that of a PCB adduct.



of the spectra of bile pigments. There are three methyls at C-7, -13, and -17 singlets

resonating at 2.13(2.13), 2.11(2.11) and 2.02(2.02) ppm. The ethyl group at C-18, is

seen as a multiplet at 2.24(2.24) ppm (18- CH2CH3) and a triplet at 1.01(1.01) ppm

(18-CH2CH3). The multiplet resonances from the methylene groups of the propionic

acid side chains at C-8 and C-12 were found at 2.90(2.90) and 2.36(2.36) ppm. The

α-CH2 and β-CH2 of mercaptoethanol resonate at 3.64(3.64) and 2.87(2.87) ppm,

thus confirming, the binding via the S-atom. There are major chemical shift difference

(0.13 ppm) of the 31-CH3 protons, and several smaller differences (< 0.05 ppm) of


other respective signals between iPCB-ME 29 and iPCB-ME 31. Based on these

datas, we propose the following structure of iPCB-ME 29 and iPCB-ME 31 (Fig. 3-70).

They are isomeric PCB-ME addition product, with different relative stereochemistries

at C-2 and C-31.

Table 3-25: 1H NMR (600-MHz) assignments for iPCB-ME 29 and iPCB-ME 31 (10 mM TFA/D2O).

NH

NH HN

COOHCOOH

HN

OOH

A

BC

D

12

34

5

6

1819

17

16

15

14

31

7

89

101112

13

S

CH2

CH2

OH

**

FIG. 3-70: Structure of iPCB-ME 29 and iPCB-ME 31 (bound 2,22-DBV) , * indicate asymmetric C-atoms

Chemical shift (ppm) iPCB-ME 29 iPCB-ME 31


Number of H’s

Assignment

6.94 6.94 s 1 10-H 6.30 6.30 s 1 15-H 5.89 5.73 s 1 5-H

4.06 4.06 q 6.92 1 3’-H 3.64 3.60 t 6.23 1 ME α-CH2 3.42 3.44 m 1 2-H 2.90 2.90 m 4 8,12-CH2CH2COOH 2.87 2.87 m 2 ME β-CH2 2.36 2.36 m 4 8,12-CH2CH2COOH 2.24 2.24 m 2 18- CH2CH3 2.13 2.13 s 3 17-CH3 2.11 2.11 s 3 13-CH3 2.02 2.01 s 3 7-CH3 1.38 1.25 d 6.98(7.47) 3 3’- CH3 1.21 1.17 d 6.91(6.96) 3 2-CH3 1.01 1.01 t 3 18- CH2CH3


3.7.4 Chromophore transfer from CpcS-PCB to ME

In order to clarify the function of CpcS in the formation of the ME products, we used

the weak fluorescence of CpcS-PCB as compared to that of the CpcS-PCB adduct.

When CpcS-PCB and mercaptoethanol were mixed, the ensuing absorption and

fluorescence spectra (Fig.3-71) led to a final state with spectra that were nearly

identical to those in Fig. 3-62. This indicates that PCB was transferred from

CpcS-PCB to mercaptoethanol, and formed a CpcS-iPCB-mercaptoethanol complex

(CpcS-iPCB-ME).

0

50

100

150

200

250

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.0

0.2

0.4

0.6

1 min 30 min 60min 120min 180min 240min

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

FIG. 3-71: Chromophore transfer from CpcS-PCB to mercaptoethanol. Absorption (left) and fluorescence (right) after mixing of CpcS-PCB (10μM) and mercaptoethanol (6 mM). The first spectrum was recorded 1 min after mixing, the subsequent ones every 30-60 min up to 240 min. Sample was maintained at 35 °C throughout.

3.7.5 Chromophore transfer from bilin adducts to CpcB(C155I)

3.7.5.1 Chromophore transfer from CpcS-iPCB-ME to CpcB(C155I)

Incubation of CpcS-iPCB-ME with CpcB(C155I) resulted in an increased and red

shifted absorption (from 599 to 618 nm) and fluorescence emission (from 630nm to

645 nm) (Fig. 3-72). The reaction was nearly complete in 3 hr. The spectral features

of final product after 3 hr are similar that of PCB-CpcB(C155I) (Zhao et al. 2006a),

indicating that iPCB was isomered and transferred from CpcS-iPCB-ME to

CpcB(C155I), and formed PCB-CpcB(C155I).


600 650 700 750 8000

100

200

300

400

500

600

700 1 min 30 min 60 min 120 min 180 min 240 min

Fluo

resc

ence

[a.u

.]

Wavelength [nm]300 400 500 600 700 800

0.0

0.1

0.2

0.3

0.4

0.5

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

1 min 30 min 60 min 120 min 180 min 240 min

FIG. 3-72: Chromophore transfer from CpcS-iPCB-ME to CpcB(C155I). Absorption (left) and fluorescence (right) after mixing of CpcS-iPCB-mercaptoethanol (2 μM) with CpcB(C155I) (10 μM). The first absorption and fluorescence spectra were recorded 1 min after mixing, the subsequent ones every 30-60 min up to 240 min. Sample was maintained at 35 °C throughout. Arrows indicate the spectral changes during the reaction.

3.7.5.2 Chromophore transfer from iPCB-ME to CpcB(C155I)

Next, we tested if the chromophores can be transferred from the isolated iPCB-ME

and PVB-ME adducts to cysteine-84 of the acceptor protein, CpcB(C155I). First,

iPCB-ME 29 and 31, respectively, were incubated with both CpcS and CpcB(C155I).

The ensuing spectral chances are shown in Fig. 3-73. The absorption maximum of

iPCB-ME increased and shifted significantly from 598 to 618 nm, the reaction was

nearly complete in 3hr. Incubation of PCB-ME, CpcS and CpcB(C155I) also resulted

in the induction of a red fluorescence with an emission maximum at 642 nm. The

spectral features of final product are similar that of PCB-CpcB(C155I) (Zhao et al.

2006a), indicating that iPCB was transferred, with concomitant isomerization to PCB,

from iPCB-ME to CpcB(C155I), and formed PCB-CpcB(C155I).

0

50

100

150

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.04

0.08

0.12

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

50

100

150

200

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

A B

FIG. 3-73: Chromophore transfer from iPCB-ME to CpcB(C155I). Absorption (left) and fluorescence (right) after mixing of iPCB-ME 29(A) or iPCB-ME 31 (B) (2 μM), CpcS (10 μM) with CpcB(C155I) (10 μM). The absorption was recorded 1 min (—) and 3hr (…) after mixing, the fluorescence spectrum was recorded 3hr after mixing. Sample was maintained at 35 °C throughout.


3.7.5.3 Chromophore transfer from PVB-ME to CpcB(C155I)

Last, PVB-ME 27 and 28, were incubated individually with both CpcS and

CpcB(C155I). The ensuing final spectra after 3 hr are shown in Fig. 3-74. The

absorption maximum of PVB-ME increased significantly and shifted from 556 to 550

nm, and an intense fluorescence (λmax= 588 nm) arose simultaneously. The spectral

features of final product were reminiscent of α-PEC in the Z-form, indicating that also

the PVB chromophore is transferred to the apoprotein by CpcS.

0

100

200

300

400

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

0

50

100

150

Fluo

resc

ence

[a.u

.]

300 400 500 600 700 8000.00

0.04

0.08

0.12

0.16

Abs

orpt

ion

[a.u

.]

Wavelength [nm])

A B

FIG. 3-74: Chromophore transfer from PVB-ME to CpcB(C155I). Absorption (left) and fluorescence (right) after mixing of PVB-ME 27 (A) or PVB-ME 28 (B) (2 μM), CpcS (10 μM) with CpcB(C155I) (10 μM). The absorption was recorded 1 min (—) and 3hr (…) after mixing, the fluorescence spectrum was recorded 3hr after mixing. Sample was maintained at 35 °C throughout.

3.7.6 Discussion

PCB binds to CpcS and ME, forms CpcS-iPCB-ME complexes with the novel iPCB

chromophore, which can then be transferred with concomitant re-isomerization to the

acceptor apoprotein CpcB(C155I). The intermediate complex was digested with

trypsin and then analyzed by HPLC; it yields two isomeric iPCB-ME addition products.

CpcS catalyses transfer of the chromophore of both complexes to the acceptor

apoprotein CpcB(C155I), again with concomitant re-isomerization to PCB, to yield

PCB-CpcB(C155I). PCB binds to ME also auto-catalytically, in this case four products

could be isolated by HPLC that contain ME. Two of them are identical to the

aforementioned iPCB-ME complexs, two contain a PVB chromophore. CpcS

catalyses not only transfer of the iPCB, but also of the PVB chromophore to the

acceptor apoprotein CpcB(C155I), to yield PCB-CpcB(C155I) and PVB-CpcB(C155I)

respectively. Based on these experimental results, we propose the following reaction


scheme among CpcS, PCB, CpcB(C155I) and ME (Fig. 3-75) .

CpcSCpcB(C155I)

PVB-CpcB(C155I)PVB-ME

PCB CpcS-iPCB-ME PCB-CpcB(C155I)

iPCB-ME

CpcB(C155I)CpcS+ME

ME

ME

CpcS+ME

ME

Trypsin

FIG. 3-75: Reaction of the CpcS/PCB/CpcB(C155I)/ME system.

Formation of the iPCB-ME and PVB-ME adducts is again principally an “artifact” due

to the presence of mercaptoethanol in the reaction mixture. Chromophore attachment

catalyzed by CpcS does not require a thiol as cofactor, unlike the catalytic activity of

the α84-lyase, CpcE/F. However, their formation indicates that PCB can covalently

binds to thiols without additional catalysts, and thereby possibly also to a cysteine

residue of the lyase, or the acceptor protein. Moreover, none of the products is a true

PCB adduct that contains the 2H,3H-PCB chromophore; instead two pairs of products

are formed that contain isomerized chromophores, viz, iPCB or PVB. Autocatalytic

addition to the acceptor protein is known in the classical and in the new type of

phytochromes where the binding cysteine is near aa 250 and the N-terminus,

respectively (Lamparter et al. 2004), and in ApcE from Anabaena sp. PCC 7120

(Zhao et al., 2005). In the latter cases, the bound chromophore is PCB. PecA adds

autocatalytically PCB, but this reaction is of low fidelity compared to lyase-catalyzed

reaction. It yields a low absorbance, low fluorescence PCB-PecA adduct, its

absorption maximum is at λ=645 nm (Boehm et al., 2007). The covalent binding of

ME to PCB, yielding iPCB-ME, may relate to these observations, such that the thiol

binds first to the chromophore, and this then promotes transfer of the chromophore to

another amino acid, including cysteine. Judged from the absorption and NMR spectra


of iPCB-ME 29, 31, the chromophore is bound in the two products via the 31-C, and

can be transferred to Cys of apoproteins. This is an indication that such intermediary

addition products may also occur during the enzymatic reaction which involves

nucleophilic addition to the ethylidene group, either of cofactor, or of a suitable amino

acid, including in particular imidazoles and cysteines.

The heterodimeric lyase/isomerase (PecE/PecF) catalyzes both the covalent

attachment of PCB and the concurrent isomerization of the molecule to PVB, yielding

PecA-PVB (see 3.1). The heterodimeric lyase/isomerase (CpcE/CpcF) catalyzes the

covalent attachment of PCB, yielding CpcA-PCB (see 3.2). These two α-apoproteins

(CpcA and PecA) from Anabaena PCC7120 are highly homologous (75% similar,

58% identical). The relative lyase’s homologies of CpcE and PecE (59% similar, 48%

identical), CpcF and PecF (49% similar, 31% identical) are relatively low. There is a

question how to explain the different roles of heterodimeric lyases, or what is the

catalytic mechanism of heterodimeric lyase. Both require thiols (Fairchild et al., 1992;

Zhao et al., 2000) in the in vitro reconstitution, and there are also thiols present in the

E. coli cells. It is conceivable from our results that these thiols function as the

nucleophiles that transiently add to the chromophore, yielding the 2,22H-adduct

(iPCB). Depending on the lyases, this chromophore then isomerizes either to the PCB

(=2,3H) or the PVB (=4,5H) chromophore during transfer to the apoproteins, CpcA

and PecA, respectively. This idea is summarized in the following scheme (Fig. 3-76).

Free PCB

Bound 2,22-DBV(iPCB)

Bound 4,5-DBV(PVB) Bound 2,3-DBV(PCB)

nucleophile

Transfer and isomerization

CpcE/FPecE/F

FIG. 3-76: Minimum mechanism for chromophore transfer and isomerization


3.8 Attachment of a locked chromophore, 15Za-PCB

3.8.1 Structure of 15Za-PCB

The locked chromophore, 15Za-PCB, was a generous gift of Prof. K. Inomata

(Kanazawa University, Japan). The structure of 15Za-PCB is shown in Fig. 3-77.

FIG. 3-77: Structure of 15Za-PCB

3.8.2 Absorption and extinction coefficient of 15Za-PCB

We measured absorption of 15Za-PCB under different conditions (Fig. 3-78). The

spectrum of the free 15Za-PCB in KPP (pH 7.5) is structured, with the main band

around 580 nm and an intense shoulder around 680 nm. In acidic MeOH, the red

absorption is slightly blue-shifted (λmax ≈ 680nm), but has only a low extinction

coefficient (ε678= 18,200 M-1cm-1), compared with the absorption and extinction

coefficient of free PCB ( λmax ≈ 690nm , ε690=37,900 M-1cm-1) (Cole et al., 1967). This

indicates that the locked conformation of 15Za-PCB is unlike the one assumed by

PCB after protonation.


300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

FIG. 3-78: Absorption spectra of free 15Za-PCB in acidic MeOH (—) and neutral KPP (pH=7.5, ----)

3.8.3 Chromophore binding assay

3.8.3.1 Complex formation of 15Za-PCB with CpcS

When CpcS and 15Za-PCB were mixed, they quickly formed a complex. Already the

first spectrum taken “immediately” after mixing (~1 min) had changed markedly. The

absorption had shifted from 579 to 632 nm and there is a marked fluorescence at λmax

≈680nm (Fig.3-79, 3-78), both then remained stable. These spectral changes indicate

strong interactions between CpcS and 15Za-PCB. The complex 15Za-PCB-CpcS is

only weakly fluorescent, indicating some conformational flexibility of the bound

chromophore. The absorption of 15Za-PCB-CpcS is similar of the PCB-CpcS (λmax

=634nm), while fluorescence spectrum had red shifted from 630nm to 680nm (section

3.6).


300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

15Za-PCB+CpcS 1 min 30 min 60 min 90 min 120 min

632nm

600 650 700 750 8000

5

10

15

20

Fluo

resc

ence

[a.u

.]

Wavelength [nm]

15Za-PCB+CpcS 1 min 30 min 60 min 90 min 120min

FIG. 3-79: Binding of 15Za-PCB to CpcS. Absorption (left) and fluorescence (right) changes on formation of 15Za-PCB-CpcS. Spectra of 15Za-PCB (5 μM) in KPB (500 mM, pH 7.5) containing NaCl (100 mM) plus CpcS (15 μM) recorded 1 min after mixing, this spectrua remained unchanged for 2 hr.

3.8.3.2 Spontaneous binding of 15Za-PCB with CpcB(C155I)

When CpcB(C155I) and 15Za-PCB were mixed, the resulting absorption and

fluorescence changed very fast in 1 min and slowly increase over a period of 2hr

(Fig.3-80). The complex formation of 15Za-PCB-CpcB(C155I) is kinetically similar to

the spontaneous formation of “aberrant” PCB-CpcB(C155I) (with incorrectly attached

chromophore). The formed 15Za-PCB-CpcB(C155I) has a main absorption band

around 660 nm, a weak shoulder around 700 nm and a fluorescence band around 670

nm; this is quite different from “aberrant” PCB-CpcB(C155I), which absorbs at

λmax≈640 nm.

600 650 700 750 8000

20

40

60

80

100

120

140

Fluo

resc

ence

[a.u

.]

Wavelength [nm]

15Za-PCB+CpcB(C155I) 1 min 30 min 60 min 90 min 120min

670nm

300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

0.25

0.30

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

15Za-PCB+CpcB(C155I) 1min 30 min 60 min 90 min 120 min

662nm

699nm

FIG. 3-80: Binding of 15Za-PCB to CpcB(C155i). Absorption (left) and fluorescence (right) changes on formation of 15Za-PCB-CpcB(C155I) after mixing 15Za-PCB (5μM) and CpcB(C155I) (15 μM) in KPB (500 mM, pH 7.5) containing NaCl (100 mM).


3.8.3.3 15Za-PCB binding to CpcB(C155I) catalyzed by CpcS

When CpcB(C155I), CpcS and 15Za-PCB were mixed, the resulting absorption and

fluorescence changes were like a superposition of the changes occurring with CpcS

and CpcB(C155I) individually. The first spectra obtained 1 min after mixing was like

that shown in Fig. 3-79, over the next two hrs the spectra then changed to those

observed with CpcB(C155I). (Fig.3-81). Obviously, the reaction first produced an

intermediate 15Za-PCB-CpcS (see 3.8.3.1), then the chromophore is transferred from

15Za-PCB-CpcS to CpcB(C155I), forming 15Za-PCB-CpcB(C155I). It indicates that

the formation of 15Za-PCB-CpcB(C155I) is slower than the formation of

15Za-PCB-CpcS. The formation of 15Za-PCB-CpcB(C155I) has a main absorption

band around 660 nm, a weak shoulder around 700 nm and a fluorescence band

around 670 nm. These spectral features are the same as that of the spontaneous

addition product (3.3.8.2). It indicated that the formation of 15Za-PCB-CpcB(C155I)

was autocatalyzed , and did not need the CpcS lyase. This is a remarkable difference,

compared with the formation of native PCB-CpcB(C155I), which absorbs at λmax= 619

nm (section 3.4) and has a stronger fluorescence.

600 650 700 750 8000

20

40

60

80

100

Fluo

resc

ence

[a.u

.]

Wavelength [nm]

15Za-PCB+CpcS+CpcB(C155I) 1 min 30 min 60 min 90 min 120min

668nm

300 400 500 600 700 8000.00

0.05

0.10

0.15

0.20

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

15Za-PCB+CpcS+CpcB(C155I) 1 min 30 min 60 min 90 min 120 min

662nm

FIG. 3-81: Binding of 15Za-PCB to CpcB(C155I) in the presence of CpcS. Absorption (left) and fluorescence (right) changes on formation of 15Za-PCB-CpcB(C155I) after mixing 15Za-PCB (5 μM) , CpcB(C155I) (15 μM) and CpcS (15 μM) in KPB (500 mM, pH 7.5) containing NaCl (100 mM).

3.8.4 Denaturation of the 15Za-PCB addition products

When we denature the adduct 15Za-PCB-CpcS with 8 M urea, the spectra at pH 7 or

pH 1.5 are identical to the respective spectra of the free chromophore (Fig. 3-82, right).


It indicates that the 15Za-PCB is not bound covalently to the lyase CpcS. If, however,

the adduct 15Za-PCB-CpcB(C155I) is denatured, the spectra at pH 7 or pH 1.5 are

different to the respective spectra of the free chromophore (Fig. 3-82, left). The

blue-shift of ~20 nm, as compared to the free chromophore, is compatible with the

loss of one conjugated double bond, this is compatible with an addition to the

3-ethylidene group of 15Za-PCB and formation of a covalent bond to the apoprotein

CpcB(C155I).

300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

15Za-PCB-CpcB(C155I) native pH7 denative pH7 denative pH1.5

662nm

668nm

659nm

607nm

610nm

300 400 500 600 700 8000.0

0.1

0.2

0.3

Abso

rptio

n [a

.u.]

Wavelength [nm]

CpeS-15Za-PCB native pH7 denative pH7 denative pH1.5

679nm

617nm

629nm

FIG. 3-82: Absorption of the denatived 15Za-PCB-CpcB(C155I) (left) and 15Za-PCB-CpcS (right) in different conditions

3.8.5 Chromophore reaction with Zn2+

The resulting fluorescence changed markedly during the formation of chromophore

addition products. These complexes were further characterized by Zn2+ fluorescence

staining. To detect the effect of Zn2+, we first tested the reaction of the free

chromophore with Zn2+. When 15Za-PCB and 1.5 M Zn-acetate were mixed, the

absorption decreased and broadened over a period of 30 min, and a weak

fluorescence nearly reached saturation within 10 min (Fig.3-83). There is already a

double-peaked intermediate after 1 min that is almost as fluorescent as the final

product. The results of PCB reaction with Zn-acetate showed that the absorption of the

intermediate (1 min) and after 30 min is different, and the fluorescence at shorter

wavelength (Fig. 3-84). It indicated that the pigment can form a Zn-complex that has a

weak fluorescence. These differences probably reflect different conformations of the

two chromophores.


550 600 650 700 750 8000

20

40

60

15Za-PCB+1.5M Zn(CH3COO)2

1 min 10 min 20 min 30 min

Fluo

resc

ence

[a.u

.]

Wavelength [nm]300 400 500 600 700 800

0.00

0.01

0.02

0.03

0.04

0.05

0.06A

bsor

ptio

n [a

.u.]

Wavelength [nm]

15Za-PCB+1.5M Zn(CH3COO)2


FIG. 3-83: Binding of 15Za-PCB to Zn2+. Absorption (left) and fluorescence (right) changes on formation of 15Za-PCB-Zn2+ after mixing 15Za-PCB (5 μM) in 1.5 M Zn-acetate (pH=5.6) Compare Fig. 3-78 for the spectra of the free chromophore.

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

0.12

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

PCB+1.5M Zn(CH3COO)2

pH 5.6 1 min 10 min 20 min 30 min 40 min

618nm

550 600 650 700 750 8000

20

40

60

80

100PCB+1.5M Zn(CH3COO)2

pH 5.6 1 min 10 min 20 min 30 min 40 min

Fluo

resc

ence

[a.u

.]

Wacelength [nm]

629nm

FIG. 3-84: Binding of PCB to Zn2+. Absorption (left) and fluorescence (right) changes on formation of PCB-Zn2+ after mixing PCB (5 μM) in 1.5 M Zn-acetate (pH=5.6).

3.8.6 The effect of pH on chromophore spectra

As stated in section 3.8.3.2, the CpcB(C155I) adduct with 15Za-PCB absorbs quite

differently than the adduct with PCB. Since one reason might be a differential

protonation, bilin spectra are strongly pH-dependent. We therefore did a pH-titration of

the locked chromophore 15Za-PCB, in parallel with PCB (Fig. 3-85). It indicates that

the pKa is shifted from ~6 (PCB) to ~5 (15Za-PCB). The pKb of PCB (~10) is not seen

in this titration, but the changes with 15Za-PCB at high pH indicate that the pKb is also

shifted to lower pH. Maybe incomplete protonation is then reason for the low ε of

15Za-PCB in acidic methanol (see section 3.8.2). Since this difference may be


relevant in the protein bound situation, one reason for different adduct spectra with

CpcB(C155I) is possibly that 15Za-PCB is not protonated; PCB is protonated in all

native structures (Sawatzki et al., 1990).

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.50.02

0.03

0.04

0.05

0.06

Abs

orpt

ion@

590n

m [a

.u.]

pH

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.50.02

0.04

0.06

0.08

Abs

orpt

ion@

685n

m [a

.u.]

pH

FIG. 3-85: The effect of pH on PCB (left) and 15Za-PCB (right) absorption( 500mM KPP).

3.8.7 Conclusion

PCB and the conformationally restricted 15Za-PCB have the following different

characters.

There are distinct differences in the absorption, extinction coefficient in acidic

methanol and pKa of the free 15Za-PCB, compared with that of free PCB.

Similar to PCB, 15Za-PCB is not bound covalently to the lyase CpcS, and bound

covalently to the apoprotein CpcB(C155I), judged for their denatured absorption

spectra. However, autocatalytic binding gives spectroscopically the same product as

binding catalyzed by CpcS, The absorption of 15Za-PCB-CpcS is similar of that of

PCB-CpcS, while fluorescence is red shifted by 50 nm.

Two pigments can form Zn-complexes, they have different absorption and

fluorescence.


3.9 Test of a protein with Heat-like repeats, DOHH, for

lyase activity

DOHH (deoxyhypusine hydroxylase) has a Heat-like repeats protein from the malaria

parasite, Plasmodium falciparum (Kaiser et al., 2006, 2007), it completes hypusine

biosynthesis through hydroxylation. These repeats are characteristic for the E/F-type

biliprotein lyases. DOHH is, in particular, distantly homologous to CpcE, we therefore

tested the chromophore-attaching activity using the binding assay of PCB binding to

PecA in the presence of DOHH (gift of Prof. A. Kaiser (University Siegburg-Bonn,

Germany). None of the experiments (DOHH replacing PecE and PecF (Fig. 3-86), or

only PecF (Fig. 3-87) or PecE (Fig. 3-88) did result in any significant changes,

indicating that DOHH has no bilin lyase activity. The small changes, in particular a

shoulder around 420 nm, are possibly due residual interactions, or to unspecific thiol

binding to C-10 and formation of rubinoid pigment(s) with other factors in the

expression system.

300 400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

PecA+PCB PecA+PCB+DOHH

600 700 8000

100

200

300

400

Fluo

resc

ence

[a.u

.]

Wavelength [nm]

PecA+PCB PecA+PCB+DOHH

FIG. 3-86: Effect of DOHH on binding of PCB to PecA. Absorption (left) and fluorescence (right) of PCB-PecA after mixing PCB and PecA with, or without, DOHH in the reconstitution system (Tab. 2-1)

600 700 8000

50

100

150

200

Fluo

resc

ence

[a.u

.]

Wavelength [nm]

PecA+PCB+PecE PecA+PCB+PecE+DOHH

300 400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

PecA+PCB+PecE PecA+PCB+PecE+DOHH

FIG. 3-87: Effect of DOHH on binding of PCB to PecA. Absorption (left) and fluorescence (right) of PCB-PecA after mixing PCB, PecA and PecE with, or without, DOHH in the reconstitution system (Tab. 2-1)


300 400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

Abs

orpt

ion

[a.u

.]Wavelength [nm]

PecA+PCB+PecF PecA+PCB+PecF+DOHH

FIG. 3-88: Effect of DOHH on binding of PCB to PecA. Absorption of PCB-PecA after mixing PCB, PecA and PecF with, or without, DOHH in the reconstitution system (Tab. 2-1)

We therefore repeated the experiment of PCB binding to PecA in the presence of high

concentration DOHH supernatant, in parallel with an “empty” cell supernatant (Fig.

3-89). The result shows that there is a difference in the 423 nm region, and DOHH

and control supernatant both have this absorption band. It indicates that the difference

is not due to DOHH, and since there is no change in the region >500 nm, we conclude

that DOHH is inactive as bilin lyase.

300 400 500 600 700 800

0.00

0.05

0.10

0.15

0.20

Abs

orpt

ion

[a.u

.]

Wavelength [nm]

PecA+PCB PecA+PCB+control PecA+PCB+DOHH

423nm

FIG. 3-89: Effect of DOHH on binding of PCB to PecA. Absorption of PCB-PecA after mixing PCB and PecA with, or without, DOHH/contrrol in the reconstitution system (Tab. 2-1)

Based on above experiments, DOHH has no chromophore-attaching activity. Since

Heat-like repeats are believed to be responsible fro protein-protein interactions rather

than for catalytic activities, it is likely that they have this function, too, in DOHH.

4. Concluding Discussion 136

4: Concluding Discussion 4.1 A multi-plasmidic expression system

The first reconstitution system for a biliprotein, holo-CpcA, was developed by

Tooley et al. (2001). We also established a multi-plasmidic expression system for

reconstitution of phycobiliproteins in E. coli. After cloning of apophycobiliprotein

genes, phycobilin biosynthesis genes and lyase genes from several cyanobacteria,

various phycobiliproteins could be biosynthesized in the heterologous E. coli

system using dual plasmids containing their respective genes. CpcA, PecA and

subunits of allophycocyanin from Anabaena PCC7120 as well as C84-PCB and

C155-PCB of PecB and CpcB from M. laminosus were successfully prepared by

this way.

This heterologous system in E. coli is more advantageous than reconstitution in

vitro in the following three respects: (1) On reconstitution in vitro, where the lyase

and acceptor genes were first overexpressed in E. coli, and then reconstituted in

vitro with isolated phycobilins, the lyases and/or the apoproteins were often difficult

to dissolve and refold. Reconstitution yields in vitro were therefore often low and

poorly reproducible, and even in favorable cases the system had to be optimized

individually for each biliprotein. An example is the phycoerythrin reconstitution

(Zhao et al., 2007a). Such problems are largely avoided with the E. coli system, in

particular if expressions are done at low temperatures (16 – 20 °C). (2) In vitro

reconstitution is complicated by the fact that most apo-phycobiliproteins bind

phycobilins spontaneously, but with low fidelity and often with low yield. This results

in unwanted products and mixtures that are difficult to analyze and handle, and

complicate enzymatic studies. As shown by controls without lyase genes,

spontaneous addition is, in E. coli, generally at or below the detection limits. (3) The

system allows, at least in principle, the in vivo labeling of fusion products of

phycobiliproteins with target proteins. A disadvantage of the system is that

enzymatic and mechanistic studies are more difficult. However, for such work

carefully chosen pairs of “well behaving” lyase(s) and acceptor proteins can be

used as case studies. The E. coli reconstitution system also has some potential

problems like imbalanced expressions, incompatibility of plasmids and low yields,


but this was generally no problem, and, in case, could be successfully managed.

Recently, Guan et al. (2007) successfully biosynthesized α-PC in E. coli by using a

single expression vector. The applied vector, pCDFDuet , harbored two cassettes:

one cassette carried the genes hox1 and pcyA required for conversion of heme to

phycocyanobilin (PCB), and the other cassette carried cpcA encoding CpcA along

with the lyase genes cpcE and cpcF. The work showed that one vector can express

five genes. There were four vectors (pACYADuet, pCDFDuet, pCOLADuet,

pETDuet) included in our multi-plasmidic expression system. It is expected that this

multi-plasmidic expression system can principally express up to 20 genes, which

may suffice for a simple phycobilisome including ApcA,B,C,D,E,F; CpcA,B; HO1,

PcyA, CpcE,F, CpcS,T; LCR, L30R, L8

R; methylase(s) (Miller et al., 2008; Shen et al.,

2008b) and methionine-peptidase(s). It seems feasible that, on this basis, one

could now produce in E. coli fully matured integral biliproteins like APC, CPC, or

even simple phycobilisomes

In extending this methodology to other systems, the expression in other prokaryotic

or in eukaryotic cells of an appropriate selection of genes encoding enzymes and

apophycobiliprotein subunit-containing fusion proteins could lead to the intracellular

production of constructs carrying specific bilins at unique locations, with broad utility

in addressing a variety of questions in cell biology.

4.2 A multi-step analytic system

We also established a multi-step analytical system to analyze the reconstituted

phycobiliproteins. The system includes the following five important steps.

1) Phycobiliproteins, apoprotein and lyase with His-tag were purified by using a

combination of chromatographic techniques. A quick enrichment of the His6-tagged

biliproteins is possible by Ni2+-affinity chromatography. For better purification, it was

followed by fast Performance Liquid Chromatography (FPLC) and ion exchange

(DEAE) chromatography (Zhao et al., 2002).

2) Quantitative absorbance, fluorescence, circular dichroism spectra of the

products were recorded, and compared with those of the natural subunits of various


phycobiliproteins from several cyanobacteria. The chromophore was further

characterized by using the same techniques on the denatured biliproteins. Covalent

binding was established by SDS-PAGE followed by Zn2+ fluorescence staining

(Berkelman and Lagarias, 1986;Tooley et al., 2001,2002; Parbel et al., 1997;

Nomsawai et al., 1999).

3) Proteolytic digestion of purified reconstituted phycobiliproteins with trypsin or

pepsin. The digested samples were purified by P-6 DG (Bio-Rad) column and

Sep-Pak C18 cartridge, and then fractionated by HPLC on reverse phase C18

column and detection of chromopeptides with diode array detection (Swanson and

Glazer, 1990). This micro-preparative system without KPP is suitable for preparing

samples for MS and NMR spectroscopy.

4) Tryptic chromopeptides were analyzed by mass spectrometry in positive ion

mode with a nano-ESI source. Sequences of these peptides could be found when

MS–MS capabilities of the instrument are employed. This method is comparable in

performance to amino acid sequencing of chromophore-containing peptides (Klotz

and Glazer, 1985; Hunsucker et al., 2004), but it requires far less material, and

allows a better control of chromophore modification. There are indications that

certain chromophore types can also be distinguished by MS-MS spectroscopy

(Matthias Plöscher, private communication, 2008).

5) Peptic chromopeptides were analyzed by 1H-NMR spectroscopy (Bishop et al.,

1986, 1987), but the method was improved by using 2 mm diameter microtubes to

minimize protein requirements.

In combination, these techniques can prove the chromophore structure and the

correctness of chromophore attachment to the apoprotein in reconstituted

phycobiliproteins. Most of them have been used before, but the combination and

standardized protocols are necessary to fully characterize the products. This

multi-step analytic system is suitable to analyze native phycobiliproteins or other

reconstituted modified phycobiliproteins, and can also be extended to other

fluorescent proteins.


4.3 E/F-type lyase catalyzed attachment

The PecA-PCB and CpcA-PCB from Anabaena PCC7120, were obtained by in vivo

reconstitution. The heterodimeric lyase/isomerase (CpcE/CpcF and PecE/PecF)

catalyzes the covalent attachment of PCB at Cys-α84 of CpcA and PecA,

respectively, and the latter the concurrent isomerization of the chromophore to PVB.

Both lyase subunits are required for chromophore attachment. The reaction has

been studied in some detail with the isomerizing lyase, PecE/F from Anabaena PCC

7120. The F-subunit alone is inactive; the E-subunit alone catalyzes only the PCB

attachment without isomerization (Zhao et al., 2005, Boehm et al., 2007). We also

tested the lyase activity of the deoxyhyposyl-hydroxylase (DOHH) from the malaria

parasite, Plasmodium falciparum. This enzyme has Heat-like repeats that are

characteristic for the E/F-type lyases, but it had not chromophore-attaching activity.

PUB is another example of a chromophore that had never been found in free form

in cyanobacteria; in fact, it is probably the most abundant biliprotein chromophore

in marine cyanobacteria: Since the same ∆5-to-∆2 double-bond isomerization that

generates bound PVB from PCB would generate bound PUB from PEB, it is

reasonable to speculate that this reaction is again catalyzed by the action of an

isomerising lyase that uses PEB as substrate. A candidate has been proposed by

Six et al.(2007), but biochemical evidence for such a lyase is still lacking (Scheer

and Zhao, 2008).

4.4 S (/U)-type lyase catalyzed attachment

In the cyanobacterium Fremyella diplosiphon, a cpeCDESTR operon was sequenced

and some of the genes were shown to function in the biogenesis of phycoerythrin (PE).

The function of the cpeCDE genes and cpeR gene were identified, but the roles of the

cpeS and cpeT products were not identified (Cobley et al., 2002). Shen et al., (2004,

2008) analyzed the genome sequences of Synechococcus sp. PCC 7002 and other

strains that do not synthesize PE. This revealed paralogs, whose products displayed

very high sequence similarity to CpeS and CpeT. Paralogs of cpeS and cpeT were

present in all phycobiliprotein containing cyanobacteria, were absent in organisms

that do not synthesize phycobiliproteins, and were encoded within operons with other


phycobiliprotein-related genes including other potential lyases (CpcU and CpcV).

Based on those observations, the authors suggested that these gene products were

involved in phycobiliprotein synthesis, it seemed plausible that the CpeS and CpeT

paralogs might be phycobiliprotein lyases for PC and/or AP. Currently, the function of

CpcS from Anabaena PCC7120 as a phycobiliprotein lyase has been reported in

several publications from our laboratory, and derived in part from this work (Zhao et al.,

2007a; Zhao et al., 2006a) and of CpcS from two other cyanobacteria by the

laboratories of Bryant and Schluchter (Saunée et al., 2008; Shen et al., 2008). CpcS

(CpcS-III) from Anabaena PCC 7120 not only catalyzed PCB attachment to β-C82 of

PecB and CpcB from M. laminosus and Anabaena PCC7120, but also catalyzed PCB

attachment to all subunits of allophycocyanin from Anabaena PCC7120; it even

attached PEB to the Cys-82 position of β-PE (Zhao et al., 2007a; Zhao et al., 2006a).

Shen et al.(2008) and Saunée et al.(2008) reported the CpcS-I and CpcU comprise, in

Synechococcus sp. PCC 7002 and Synechocystis sp PCC 6803, the heterodimeric

bilin lyase that attaches phycocyanobilin to Cys-82 of β-phycocyanin and Cys-81 of

allophycocyanin subunits.

Homologs of the CpcS and CpeS proteins were classified into five groups (Shen et al.,

2008). The largest of these groups, Group C, can be further divided into three clades,

which have denoted CpcS-I, CpcS-II, and CpcS-III. CpcS from Synechococcus sp.

PCC 7002 and Synechocystis sp. PCC 6803 belong to clade CpcS-I, while CpcS from

Anabaena sp. PCC 7120 belong to clade CpcS-III. Cyanobacteria can be divided into

two groups on the basis of their PCB lyases for the Cys-82 position of β-PC.

Anabaena sp. PCC 7120 (clade CpcS-III) apparently produces lyases that are

functional as a single subunit (Zhao et al. 2006a; Zhao et al., 2007a), whereas

Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803(clade CpcS-I)

produce a heterodimeric lyase composed of a CpcS-I subunit and CpcU (Shen et al.,

2008).

CpcS is apparently a (nearly) universal lyase with respect to the protein but is highly

specific for a single binding site, cysteine-84. It is inactive, however, for the

cysteine-84-binding site of α-subunits of phycocyanin (CpcA) and PEC (PecA), which

is rather served by the site- and protein-specific EF-type lyase (Schluchter and Glazer,

1999; Zhao et al., 2000; section 3-1, 3-2). This probably indicates a structural

difference among the cysteine-84-binding sites of CpcA and PecA from other


biliproteins. This difference was supported by a sequence analysis of the apoproteins

(Zhao et al., 2007a), phylogenetic characteristics of the different types of biliproteins

(Sidler, 1994), and the N-methylation of asparagine found only in β-subunits (Klotz

and Glazer, 1987). The two groups of biliproteins may have different functions

besides the lyase specificity. The α-84 sites of all cyanobacterial and red algal

biliproteins are at the contact surface to the β-subunits in the trimers and show similar

geometric and stereochemical properties; this argues against a structural or

photosynthetic function that sets CpcA and PecA apart from all other subunits. The

difference may be relevant to phycobilisome degradation (Zhao et al., 2007a).

CpcS can bind PCB, as assayed by Ni2+ chelating affinity chromatography. Binding

is rapid, probably non-covalent, but much more strongly than with E/F-type lyases.

Tthe chromophore is bound in an extended conformation similar to that in

phycobiliproteins, but only poorly fluorescent. The extended conformation is

supported by binding studies with a conformationally locked chromophore,

15Za-PCB, which also binds rapidly and non-covalently to CpcS and gives a product

with similar spectral properties as PCB-CpcS. Upon addition of apo-biliproteins, the

chromophore is transferred to the latter much slower (~1 hr), indicating that

chromophorylated CpcS is an intermediate in the enzymatic reaction.

4.5 T-type lyase catalytic attachment

Synechococcus PCC7002 has yet another gene, cpcT, which is ubiquitous in

cyanobacteria and codes for a third family of lyases, the T-type. Recombinant CpcT

catalyzes the regiospecific PCB addition at Cys-155 of CpcB from Synechococcus

PCC7002 (Shen et al., 2006), to Cys-155 of both β-CPC and β- PEC in Anabaena sp.

PCC 7120 (Zhao et al., 2007), and to Cys-155 of both β-CPC and β- PEC from M.

laminosus (this work). From these data, CpcT is a lyase with only moderate protein

specificity that is combined with a high site-specificity.

Based on the above conclusion, the three lyases seem to be sufficient to attach all

phycobiliprotein chromophores in Anabaena sp. PCC7120, and other species

containing only APC, CPC, PEC in their PBS, but no PE: CpcS(/U) and CpcT attach

PCB to Cys-β84 and Cys-ß155, respectively, of PC and PEC, while CpcE/F and


PecE/F catalyze attachment to Cys-α84 of PC and of PEC, respectively, the latter with

a concomitant isomerization of PCB to PVB. Posttranslational modification is an

important mechanism to modulate the structural and functional properties of proteins.

Chromophore attachment is not the only post-translational modification in biliproteins.

A further post-translational modification is methylation of Asparagine-71/72 of

phycobiliprotein β-subunits (Minami et al. 1985; Klotz et al., 1986; Swanson and

Glazer, 1990; Miller et al., 2008; Shen et al., 2008b). Methylation of Asn has been

reported already in the 1980s in ß-subunit of biliproteins (Minami et al. 1985; Klotz et

al., 1986; Klotz and Glazer, 1987). Swanson and Glazer (1990) demonstrated that

methylation of this Asn residue was catalyzed by a specific methyltransferase, such a

methylase has been identified recently(Miller et al., 2008; Shen et al., 2008b).Miller et

al. (2008) conclude that this post-translational modification probably occurs after

chromophorylation but before trimer assembly in vivo. Methylated Asn-71/72 is

important for efficient energy transfer in phycobilisome and reduction of

photoinhibition at high light intensity (Swanson and Glazer, 1990; Shen et al., 2008b).

Nothing is known, to our knowledge, on the mechanism of a third post-translstional

modification, that is N-terminal methionine cleavage in several biliproteins, but such

enzymes have been characterized in other organisms (Schechter and Berger, 1967;

Miller et al., 1987; Boufous and Vadeboncoeur, 2003).

Up to date, there are three types of lyases and one methylase identified. The general

identification and characterization of a new enzyme included the following steps and

methods, which may be applied to identify new enzyme in future studies. a) Using

bioinformatics methods (e.g. comparative bioinformatics approach and gene

neighborhood analysis) analyze the various cyanobacterial genomes that have been

sequenced, find the candidate gene(s). b) Introduction of this gene into a mutant

cyanobacterial cell line, or cloning it into E.coli using the multi-plamidic ecpression

system together with other required genes. c) Culturing the wild-type and mutant cell

of the cyanobacterium, or expressing the reconstitution system in E. coli. d) Isolating

of PBS or collecting the E.coli cell. e) Protein assays and electrophoresis. f)

Absorption and fluorescence spectroscopy. g) Further analysis by CD, HPLC-MS,

NMR spectroscopy. h) Determining the kinetics and i) the catalytic mechanisms of

new enzyme. j) Studing the activity and redulation of the enzyme in the

cyanobacterium. Except for (i) and studies in the cyanobacteria, much of this has


been achieved for the three lyases.

4.6 Imidazole-bilin adducts

In the CpcS/PCB/imidazole reaction system, the amounts of the iPCB-imidazole and

adducts with oxidized (MBV) chromophore increased with imidazole concentration, at

the expense of free PCB; the amounts of the iPCB(2,22 H –bilin)-imidazole oxidation

adduct increased with temperature, at the expense of free PCB and IPCB-imidazole.

We have prepared the reaction products by HPLC, and determined their structures by

MS and NMR spectroscopy. The results showed that CpcS can promote covalent

binding of PCB to imidazole with concurrent isomerization to iPCB, and catalyses

transfer of the chromophore of the iPCB-imidazole to the acceptor apoprotein

CpcB(C155I). During this transfer reaction, the chromophore is re-isomerized to PCB,

to yield PCB-CpcB(C155I). Chromophorylation by CpcS might then involve a

histidine-bound intermediate carrying an iPCB chromophore. There is presently no

direct evidence to this in cyanobacteria, but in view of the similar absorptions of PCB

and iPCB-adducts, it requires testing using methods other than optical spectroscopy.

Conserved histidines have been found in several lyases, plant-type phytochromes and

several bacterial phytochromes (Zhao et al., 2006; Zhao et al., 2005; Wu and Lagarias,

2000; Lamparter, 2004), the reaction catalyzed by CpcS could therefore be a model

for all these reactions. We proposed the following model of chromophorylation by

CpcS (see Fig. 4-1).

Free PCB

CpcS-His-iPCB

PCB-Cys-apoprotein

apoproteinCpcS

FIG. 4-1: A catalytic mechanism model of CpcS


4.7 Mercaptoethanol-bilin adducts

In the ME and PCB reaction system, two kinds of adducts were obtained, PVB-ME

and iPCB-ME. Adducts of PCB with thiols have been described before, but

characterized only by optical spectroscopy (Köst et al., 1975). Since the optical

spectra of PCB and iPCB are rather similar, it is therefore not clear if these authors

have been working then also with iPCB adducts. The chromophores of the both types

can be transferred to CpcB(C155I), yielding CpcB(C155I)-PCB and

CpcB(C155I)-PVB, respectively. Both products are remarkable. Chromophore (iPCB

and PVB) can covalently bind to ME even in the absence of the lyase, as proved by

MS and NMR spectrum. It indicates that the autocatalytic chromophorylation might

then involve a thiol-chromophore intermediate.

Spontaneous formation of a PVB-ME adduct is intriguing, because the PVB

chromophore was before only known in its bound form to the protein. Here, it is

accessible for the first time in the (nearly) free form with only ME attached. Moreover,

it is formed in a spontaneous reaction, and its formation is inhibited in the presence of

the lyase, CpcS, indicating that one function of the lyase might be to inhibit this

irreversible reaction. This isomerization from PCB to PVB is similar to the one

catalyzed by PecF in the PecA biosynthesis. Up to now, the role of CpcF was not

identified. The PVB-ME adduct formation might explain the role of CpcF in the

holo-CpcA biosynthesis ; possibly it promotes the alternative route, that is iPCB

transfer and re-isomerization (see below), and inhibit PVB formation in the holo-CpcA

biosynthesis. This would also rationalize why homologous lyases are involved in the

attachment of PCB, which formally does not require isomerization.

The novel 2,22H-chromophore (iPCB) has been postulated first by Grubmayr et al.

(1988a,b,c), as an intermediate in a multi-step thiol addition and replacement reaction.

However, in the ME and imidazole adducts it has been prepared and identified for the

first time. Stereoisomers of the iPCB-ME adducts are the major products in the

presence of the lyase. In line with the suggestion of Grubmayr, this chromophore is

transferred in the presence of CpcS to the apoprotein, with concomitant isomerization

to the 2,3H-chromophore (PCB). It might indicate that the second function of CpcF is

to isomerize iPCB to PCB in holo-CpcA biosynthesis, and the lyase PecF might inhibit


the iPCB formation in the holo-PecA biosynthesis.

In a more qeneral sense, the formation of covalent adducts of PCB with nucleophiles

(ME) gave first indications for a mechanistic model for the lyases. The PVB-ME and

iPCB-ME adduct formation might explain the catalytic mechanism of lyases in

phycobiliprotein biosynthesis. Hugo et al. (1993) hypothesized a model reaction for

biliprotein biosynthesis, which included an important intermediate, iPCB. Our

identification of this chromophore conjugated to nucleophiles like imidazole or thiols

lends support to this hypothesis and we suggested a unified mechanistic model of

catalysis by the heterodimeric lyase CpcE/CpcF and PecE/PecF (see Fig. 3-76).

4.8 Some interesting observations as by-products of

the experiments

During the experiments, several additional interesting observations were made that

were not further explored in detail. Currently, they can not be explained well for lack of

sufficient information, and it is unclear how they fit into the complete picture of

biliprotein lyases. They are summarized here because they maybe relevant to future

studies.

1) In the Zn2+ staining assay for covalent chromophore attachment, there are different

fluorescence intensities for certain chromophores in the different Zn2+ salt solutions.

For example, the fluorescence intensity of PCB in ZnCl2 solution is higher than in Zn-

acetate solution, while the fluorescence intensity of PVB in ZnCl2 solution less than

that in Zn-acetate solution, indicating an effect of the anion on the fluorescence.

2) The adducts CpcS-PCB and ME-PCB fluoresce only weakly. However, when

CpcS-PCB and mercaptoethanol were mixed, we observed a strong fluorescence of

the ternary complex, CpcS-PCB-ME. This is typical for biliproteins, in which the

chromophore is held rigidly by strong interaction with the apoprotein. Biochemical

evidence of a covalent bond between CpcS and PCB is still lacking. It is known,

however, that such interactions can also occur in the absence of covalent binding. An

example is chromophore binding in phytochrome, where in the absence of the binding

cysteine adducts are formed that are spectrally very similar to native phytochrome,


and were even photoactive (Lamparter and Michael, 2005).

3) There are different adsorption characters of different chromopeptides on Sep-Pak

C18 cartridge. For example, the PVB and PEB chromopeptides adsorbed to Sep-Pak

C18 cartridge were eluted only with difficulties, but PCB chromopeptides were easily

eluted. This is helpful for the separation of complex mixtures containing different

chromophores. However, HPLC may therefore be problematic for chromophore

quantification under certain conditions.

4) The PVB-PecA was digested using trypsin, and separated by HPLC. We found

some products, besides the PVB-peptides, that contained the PCB, and not the PVB

chromophore. We take this as evidence that the PCB to PVB isomerization is

reversible. This has never been observed with the PecE/F lyases.

5) The chromopeptides of tryptic digestion were prepared by HPLC and measured MS.

The HPLC results shows that MS spectra of PCB- and PEB-chromopetides are very

“clean”, they gave mainly a sinlge peak in the mass spectrum. By contrast, there are

many fragment ions in the PVB- and PUB-chromopeptides. This difference indicates

that the PVB and PUB chromophores are rather labile, and easily cleaved under MS

conditions. Possibly, this can be used to distinguishing chromophore types by MS.

4.9 Future prospects

Recently, the techniques of biochemistry, molecular biology, spectroscopy, x-ray

diffraction, electron microscopy and biophysics have served to improve our

understanding of the structure of phycobilisomes, and their functions in

photosynthesis. However, it is still largely unknown how phycobilisomes are properly

assembled (and disassembled). The lyases studied here have added to our

understanding of the early processes of this assembly, in particular for PC-only

phycobilisomes, but leave a considerable number of open problems. In addition, also

some aspects of PBS structure and function activity remain to be elucidated. In my

opinion, the following problems have become obvious during this work, and can now

be tackled, using in part methods developed here:

1) In order to resolve problems of imbalanced expressions, incompatibility of


plasmids and low yields, it is essential to define optimum expression conditions and

plasmids forming of different phycobiliproteins. Establishing an on-line detection

method of phycobiliproteins would be interesting in order to analyze the kinetics.

2) Find and determine new class phycobilin lyases that specifically attaches

phycobilin to the other phycobiliproteins. Up to now, the PCB:phycobiliprotein lyases

were identified, which are, at least in principle, sufficient to attach all phycobiliprotein

chromophores in Anabaena sp. PCC7120, and other species containing only APC,

CPC, PEC in their PBS. The biggest problem is currently the biosynthesis of PEs, in

spite of evidence that CpcS may be part of the solution. Yet another problem is PUB,

which globally is probably the most abundant bilin chromophore, but the biosynthesis

is unknown.

3) Currently, E/F-type lyases (CpcE/CpcF and PecE/PecF), S-type lyases and T-type

lyase were identified, but the catalytic mechanisms for these lyases are only

incompletely understood; more work is needed to detail them further.

4) Biological self-assembly is remarkable in its fidelity and in the efficient production of

intricate molecular machines and functional materials from a heterogeneous mixture of

macromolecules. Anderson and Toole (1998) suggested a model for the assembly

pathway of the cyanobacterial phycobilisome, that is still awaiting critical testing. Up to

date, many cyanobacterial genomes have been sequenced, the molecular

architecture of several PBS has been defined, and crystal structures are available for

all major phycobiliproteins. With the identification and characterization of most of the

enzymes involved in phycobiliproteins maturation, it may become feasible to study

phycobilisome assemble in vitro using the following protocol. 1) Individual

reconstitution of all subunits of the phycobilisome with chromophores in

multi-plasmidic expression systems in E. coli. 2) Secondary modification of

chromophorylated subunits in vitro with respective enzymes. 3) Preparation of mono-

and trimers from the individual subunits in vitro. 4) Individual expression of all

linker-proteins of the phycobilisome in E. coli. Finally, trimers and linker-proteins are

incubated, yielding hopefully complete phycobilisome or phycobilisome fragments.

Alternatively one might consider to assemble a phycobilisome in one multi-plasmidic

expression system in E. coli. I believe, however, that this approach is presently very

difficult and less likely to succeed.

5. Summary 148

5: Summary Phycobilins are light harvesting pigments of cyanobacteria and red algae. In

cyanobacteria, four phycobiliproteins are organized in phycobilisomes: phycocyanin

(PC), allophycocyanin (APC), and often also phycoerythrocyanin (PEC) or

phycoerythrin (PE). Their phycobilin chromophores, linear tetrapyrroles, are

generally bound to the apoprotein at conserved positions by cysteinyl thioether

linkages. A final step in phycobiliprotein biosynthesis is the post-translational

phycobilin addition to the various biliproteins. In vivo, the correct attachment of most

chromophores is catalyzed by binding-site and chromophore-specific lyases. Only

two such lyases, which both belong to the E/F-type were known at the beginning of

this work. Two additional types, S/(U)-type and T-type lyase, have been

characterized during this work. In addition, the correct structures of the products from

all three lyase types have been verified, and evidence was obtained for the reaction

mechanisms.

This characterization relied on two methodological advances. The first is the use of a

multi-plasmidic expression system for reconstitution of phycobiliproteins in E. coli.

After cloning of apophycobiliprotein genes, phycobilin biosynthesis genes and

(putative) lyase genes from several cyanobacteria, various phycobiliproteins could

be biosynthesized in the heterologous E. coli system using dual plasmids containing

the respective genes. This heterologous system produces higher yields than the in

vitro reconstitution, it is nearly devoid of spontaneous binding, better reproducible,

and more easily controlled. The second methodological advance is the consequent

use of a combination of chromatographic, electrophoretic and spectroscopic tools

that allowed a full characterization of the structure and binding sites of attached

chromophores. This included, besides optical spectroscopy, in particular mass and

magnetic resonance (1H-NMR) spectroscopy.

Using the unmodified genes coding for both subunits of PEC, as well as their cystein

mutants, three lyases were identified for the three binding site. Besides the already

known isomerizing lyase, PecE/PecF, for Cys-84 of α-PEC, these are the two new

lyases, CpcT (all5339) for Cys-153 of β-PEC, and CpcS (alr0617) for Cys-82 of

β-PEC. The spectroscopic analysis proved that the chromophores (PCB and PVB)

5. Summary 149

are correctly attached to these three binding sites.

Similarly, three lyases were identified for the three binding sites of CPC. The well

known heterodimeric lyase (CpcE/CpcF) catalyzes the covalent attachment of PCB

to αC84 of CPC, CpcS catalyses the site-selective attachment of PCB to

cysteine-β84 in CpcB; and CpcT for cysteine-β155 of CpcB. CpcE/F is specific for

CpcA, while CpcS and CpcT can react with both CpcB and PecB. We also tested the

lyase activity of the deoxyhyposyl-hydroxylase (DOHH) from the malaria parasite,

Plasmodium falciparum. This enzyme has Heat-like repeats that are characteristic

for the E/F-type lyases, but it had not chromophore-attaching activity.

The substrate specificity of the new lyase, CpcS (coded by alr0617), was further

tested with APC subunits; It is very unspecific with regard to the acceptor protein and

attaches PCB to ApcA1, ApcB, ApcD ApcF, as well as to the product of an additional

gene, apcA2; of unknown function that is highly homologous to apcA1 coding for the

APC α-subunit. Obviously, this lyase has a much broader substrate specificity than

the E/F-type lyases, but it has high site-specificity, attaching the chromophore

exclusively to the Cys-84 (consensus sequence) binding site of the APC subunits.

CpcS from Anabaena PCC7120 is a relatively simple system, it acts as a monomer,

and does not require any cofactors. CpcS binds PCB rapidly (<1s) and relatively

strongly, but probably non-covalently. The chromophore is bound in an extended

conformation similar to that in phycobiliproteins, but only poorly fluorescent. The

extended conformation is supported by binding studies with a conformationally locked

chromophore, 15Za-PCB, which also binds rapidly and non-covalently to CpcS and

gives a product with similar spectral properties as PCB-CpcS. Upon addition of

apo-biliproteins to the PCB-CpcS (or 15Za-PCB-CpcS) complex, the chromophore is

transferred to the latter much more slowly (~1 hr), indicating that chromophorylated

CpcS is an intermediate in the enzymatic reaction.

There are distinct differences in the absorption, extinction coefficient in acidic

methanol and pKa of the free 15Za-PCB, compared with that of free PCB, which are

probably due to a shift in the pK-values by about 1 pH unit.

Nucleophilic addition products of PCB were characterized that are formed

5. Summary 150

spontaneously or by the lyases, and gave first indications for a mechanistic model for

the lyases. The first nucleophile was imidazole, which is a model for histidine. Two

imidazole-PCB adducts were prepared and the structures determined by MS and

NMR spectroscopy. Surprisingly, the chromophore is isomerized in this reaction to a

2,22 H –bilin termed iso-phycocyanobilin (iPCB). CpcS not only can promote covalent

binding of PCB to imidazole, but also catalyses the transfer of the chromophore of the

formed iPCB-imidazole to the cysteine84 of acceptor apoprotein, CpcB. During this

transfer reaction, the chromophore is re-isomerized to PCB, to yield CpcB-C84-PCB.

It indicates that chromophorylation by CpcS might then involve a histidine-bound

intermediate; this could be a model for the reaction catalyzed by CpcS.

The second nucleophile was mercaptoethanol, as a model for cysteine. In the ME and

PCB reaction system, two isomers each of isomeric PVB-ME and iPCB-ME were

obtained in a non-enzymatic reaction. The chromophore of the two complexes can be

transferred to cysteine-84 of CpcB, yielding CpcB-C84-PCB and CpcB-C84-PVB. In

the presence of the lyase, CpcS, only the iPCB adducts are formed. It indicates

autocatalytical chromophorylation might then involve a thiol-chromophore

intermediate; this could be a model for the chromophorylation reaction. At the same,

we propose a possible generalized catalytic mechanism for the non-isomerizing

heterodimeric lyase, CpcE/CpcF, and its isomerizing homolog, PecE/PecF.

6. References 151

6: References

Adir N. (2005) Elucidation of the molecular structures of components of the phycobilisome : reconstructing a giant. Photosynth. Res. 85:15-32

Adir N, Dobrovetsky Y, Lerner N. (2001) Structure of c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus at 2.5 A: structural implications for thermal stability in phycobilisome assembly. J. Mol. Biol. 313:71-81.

Adir N, Vainer R, Lerner N. (2002) Refined structure of c-phycocyanin from the cyanobacterium Synechococcus vulcanus at 1.6 A: insights into the role of solvent molecules in thermal stability and co-factor structure. Biochim. Biophys. Acta. 1556:168-174.

Ajlani G. and Vernotte C. (1998) Deletion of the PB-loop in the LCM subunit does not affect

phycobilisome assembly or energy transfer functions in the cyanobacterium Synechocystis sp. PCC6714. Eur. J. Biochem. 257: 154-159

Ajlani G, Vernotte C. (1998) Construction and characterization of a phycobiliprotein-less mutant of Synechocystis sp. PCC 6803. Plant Mol. Biol. 37:577-580.

Alvey RM, Karty JA, Roos E, Reilly JP, Kehoe DM. (2003) Lesions in phycoerythrin chromophore biosynthesis in Fremyella diplosiphon reveal coordinated light regulation of apoprotein and pigment biosynthetic enzyme gene expression. Plant Cell 15:2448-2463.

Ammerman JW, Glover WB (2000) Continous underway measurement of microbial ectoenzyme activities in aquatic. Ecosystems. Mar Ecol Prog Ser 201:1–12

Anderson LK and Toole CM (1998) A model for early events in the assembly pathway of cyanobacterial phycobilisomes. Mol. Microbiol. 30:467-474

Apt KE, Grossman AR. (1993) Characterization and transcript analysis of the major phycobiliprotein subunit genes from Aglaothamnion neglectum (Rhodophyta). Plant Mol Biol.21:27-38.

Apt KE, Collier JL, Grossman AR. (1995) Evolution of the phycobiliproteins. J. Mol. Biol. 248: 79-96.

Apt KE, Hoffman NE, Grossman AR. (1993) The gamma subunit of R-phycoerythrin and its possible mode of transport into the plastid of red algae. J Biol Chem. 268:16208-16215.

Araoz R, Lebert M, Hader DP. (1998) Electrophoretic applications of phycobiliproteins.

6. References 152

Electrophoresis. 19:215-219.

Araoz R, Shelton M, Lebert M, Hader DP. (1998) Differential behaviour of two cyanobacterium species to UV radiation. Artificial UV radiation induces phycoerythrin synthesis. J Photochem Photobiol B. 44:175-183.

Arciero DM, Bryant DA, Glazer AN. (1988a) In vitro attachment of bilins to apophycocyanin. I. Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C- phycocyanin. J. Biol. Chem. 263:18343-18349.

Arciero DM, Dallas JL, Glazer AN.(1988b) In vitro attachment of bilins to apophycocyanin. II. Determination of the structures of tryptic bilin peptides derived from the phycocyanobilin adduct. J. Biol. Chem. 263:18350-18357.

Ashby MK, Mullineaux CW. (1999) The role of ApcD and ApcF in energy transfer from phycobilisomes to PS I and PS II in a cyanobacterium.Photosynthe Res. 61:169-179.

Balabas BE, Montgomery BL, Ong LE, and Kehoe DM. (2003) CotB is essential for complete activation of green light-induced genes during complementary chromatic adaptation in Fremyella diplosiphon. Mol Microbiol. 50: 781–793.

Barber J, Morris EP, da Fonseca PC. (2003) Interaction of the allophycocyanin core complex with photosystem II. Photochem Photobiol Sci. 2 :536-541.

Batard P, Szollosi J, Luescher I, Cerottini JC, MacDonald R, Romero P. (2002) Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations. Cytometry. 48:97-105.

Beale SI, Cornejo J. (1991) Biosynthesis of phycobilins. 3(Z)-phycoerythrobilin and 3(Z)-phycocyanobilin are intermediates in the formation of 3(E)-phycocyanobilin from biliverdin IX alpha. J Biol Chem. 266:22333-22340.

Beale SI, Cornejo J.(1991) Biosynthesis of phycobilins. 15,16-Dihydrobiliverdin IX alpha is a partially reduced intermediate in the formation of phycobilins from biliverdin IX alpha.J Biol Chem. 266:22341-22345.

Becker w.(2004) Microalgae in human and animal nutrition, p312-351.In Richmond, A. (ed), Handbook of microalgae culture, Blackwell, Oxford.

Berkelman TR, Lagarias JC. (1986). Visualization of bilin-linked peptides and proteins in polyacrylamide gels. Anal. Biochem. 156:194–201.

Benjamin Q. Wolfgang G. (2004) Chromophore selectivity in bacterial phytochromes: dissecting the process of chromophore attachment. Eur. J. Biochem. 271:1117-1126.

6. References 153

Bermejo R, Fernandez E, Alvarez-Pez JM, Talavera EM. (2002) Labeling of cytosine residues with biliproteins for use as fluorescent DNA probes. J. Luminescence 99:113–124.

Bhalerao RP, Lind LK, Gustafsson P. (1994) Cloning of the cpcE and cpcF genes from Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942. Plant Mol Biol. 26:313-326.

Bhat VB, Madyastha KM. (2000) C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro. Biochem Biophys Res Commun ;275:20-25.

Bhoo SH, Davis SJ, Walker J, Karniol B, Vierstra RD. (2001) Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore. Nature 414:776-779.

Bhoo SH, Hirano T, Jeong H, Lee J, Furuya M, Song P. (1997) Phytochrome photochromism probed by site-directed mutations and chromophore esterification. J Am Chem Soc 119: 11717–11718.

Bienert R, Baier K, Volkmer R, Lockau W. & Heinemann U. (2006) Crystal structure of NblA from Anabaena sp. PCC 7120, a small protein playing a key role in phycobilisome degradation. J. Biol. Chem. 281: 5216-5223.

Bishop JE, Lagarias JC, Nagy JO, Schoenleber RW, Rapoport H, Klotz AV & Glazer AN. (1986) Phycobiliprotein-bilin linkage diversity. I. Structural studies on A- and D-ring-linked phycocyanobilins. J. Biol. Chem. 261: 6790-6796

Bishop JE, Rapoport H, Klotz AV, Chan CF, Glazer AN, Flüglistaller P & Zuber H. (1987) Chromopeptides from phycoerythrocyanin: Structure and linkage of the three bilin groups. J. Am. Chem. Soc. 109: 857-881.

Björn G.S & Björn LO. (1976) Photochromic pigments from blue-green algae: phycochromes a, b and c. Physiol. Plant 36: 297-300.

Boehm S (2006) α-Phycoerythrocyanin: Dynamics of Reversible Photochromism and Lyase-catalyzed Holoprotein Assembly. Ph.D. thesis, University of Munich, Germany

Boehm S, Endres S, Scheer H, Zhao KH. (2007) Biliprotein Chromophore Attachment: Chaperone-like function of the PecE subunit of α-phycoerythrocyanin lyase. J. Biol. Chem 282:25357-25366.

Borucki B, Otto H, Rottwinkel G, Hughes J, Heyn MP, Lamparter T. (2003)Mechanism of Cph1 phytochrome assembly from stopped-flow kinetics and circular dichroism. Biochemistry 42:13684-13697.

Boufous H, and Vadeboncoeur C. (2003) Purification and characterization of the

6. References 154

Streptococcus salivarius methionine aminopeptidase (MetAP).Biochimie (Paris) 85:993–997

Brejc K, Ficner R, Huber R, Steinbacher S. (1995)Isolation, crystallization, crystal structure analysis and refinement of allophycocyanin from the cyanobacterium Spirulina platensis at 2.3 A resolution. J Mol Biol. 249:424-440.

Bryant DA. (1982) Phycoerythrocyanin and phycoerythrin: properties and occurrence in cyanobacteria. J. Gen. Microbiol. 128:835–844.

Bryant DA. (1986) The cyanobacterial photosynthetic apparatus: comparisons to those of higher plants and photosynthetic bacteria. Can Bull Fish Aquat Sci 214: 423-500

Bryant DA. (1987) The cyanobacterial photosynthetic apparatus: comparisons to those of higher plants and photosynthetic bacteria. In Platt T and Li WKW (Eds.): Photosynthetic Picoplankton. Ottawa, Canada. 214: 423-500

Bryant, DA. (1991) Cyanobacterial phycobilisomes: Progress toward complete structural and functional analysis via molecular genetics. In: Cell Culture and Somatic Cell Gentetics of Plants. Bogorad, L. & Vasil, I. K. (Eds.). pp. 257-300. Academic Press Inc., New York.

Bryant, DA. (Ed.) (1994) Advances in photosynthesis: The molecular biology of cyanobacteria. Kluwer Academic Publisher, Dordrecht.

Bryant DA, Glazer AN and Eiserling FA. (1976) Characterization and structural properties of the major biliproteins of Anabaena sp. Arch. Microbiol. 110:60–75.

Bryant DA, de Lorimier R, Guglielmi G, Stevens SE Jr. (1990)Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis. Arch Microbiol. 153:550-560.

Bryant DA, Stirewalt VL, Glauser M, Frank G, Sidler W & Zuber H. (1991) A small multigene family encodes the rod-core linker polypeptides of Anabaena sp. PCC 7120 phycobilisomes. Gene 107: 91-99.

Cai YA, Murphy JT, Wedemayer GJ, Glazer AN. (2001)Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains. Anal Biochem. 290:186-204.

Cai, YA, Schwartz SH & Glazer AN. (1997) Transposon insertion in genes coding for the biosynthesis of structural components of the Anabaena sp. phycobilisome. Photosynth. Res. 53:109–120.

6. References 155

Canaani OD, Gantt E (1980) Circular dichroism and polarized fluorescence characteristics of blue-green algal allophycocyanins. Biochemistry 19:2950–2956.

Capuano V, Braux AS, Tandeau de Marsac N, Houmard J. (1991) The "anchor polypeptide" of cyanobacterial phycobilisomes. Molecular characterization of the Synechococcus sp. PCC 6301 apce gene. J Biol Chem. 266:7239-7247.

Chang WR, Jiang T, Wan ZL, Zhang JP, Yang ZX, Liang DC. (1996) Crystal structure of R-phycoerythrin from Polysiphonia urceolata at 2.8 A resolution. J Mol Biol. 262:721-731.

Chen Fang (2006) Master thesis, HuaZhong University of Science and Technology, Wuhan, China.

Cobley JG, Clark AC, Weerasurya S, Queseda FA, Xiao JY, Bandrapali N, D'Silva I, Thounaojam M, Oda JF, Sumiyoshi T, Chu MH.(2002)CpeR is an activator required for expression of the phycoerythrin operon (cpeBA) in the cyanobacterium Fremyella diplosiphon and is encoded in the phycoerythrin linker-polypeptide operon (cpeCDESTR). Mol Microbiol. 44:1517-1531.

Cole, WJ, Chapman DJ & Siegelman HW. (1967) Structure of Phycocyanobilin. J. Am. Chem. Soc. 89: 3643-3645.

Collier JL, Grossman AR. (1992) Chlorosis induced by nutrient deprivation in Synechococcus sp. strain PCC 7942: not all bleaching is the same. J Bacteriol. 174:4718-4726.

Colyer CL, Kinkade CS, Viskari PJ, Landers JP. (2005) Analysis of cyanobacterial pigments and proteins by electrophoretic and chromatographic methods. Anal. Bioanal. Chem. 382:559-569

Cornejo J, Beale SI, Terry MJ, Lagarias JC. (1992) Phytochrome assembly. The structure and biological activity of 2(R),3(E)-phytochromobilin derived from phycobiliproteins. J Biol Chem. 267:14790-14798.

Dammeyer T, Frankenberg-Dinkel N. (2006) Insights into Phycoerythrobilin Biosynthesis Point toward Metabolic Channeling. J Biol Chem. 281:27081-27089.

Dammeyer T, Bagby SC, Sullivan MB, Chisholm SW, Frankenberg-Dinkel, N. (2008) Efficient phagemediated pigment biosynthesis in oceanic cyanobacteria.Curr Biol 18:442-448.

de Lorimier R, Wilbanks SM, Glazer AN. (1993)Genes of the R-phycocyanin II locus of marine Synechococcus spp., and comparison of protein-chromophore interactions in phycocyanins differing in bilin composition. Plant Mol Biol. 21:225-237.

6. References 156

Demidov AA, Mimuro M. (1995) Deconvolution of C-phycocyanin beta-84 and beta-155 chromophore absorption and fluorescence spectra of cyanobacterium Mastigocladus laminosus. . J Biophys. 68:1500-1506.

Debreczeny MP, Sauer K, Zhou J, and Bryant D A. (1995) Comparison of calculated and experimentally resolved rate constants for excitation energy transfer in C- phycocyanin.2.Trimers J. Phys. Chem. 99:8420–8431

Dolganov N, Grossman AR. (1999) A polypeptide with similarity to phycocyanin alpha-subunit phycocyanobilin lyase involved in degradation of phycobilisomes. J Bacteriol. 181:610-617.

Ducret A, Muller SA, Goldie KN, Hefti A, Sidler WA, Zuber H, Engel A. (1998)Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120. J Mol Biol. 278:369-388.

Ducret A, Sidler W, Wehrli E, Frank G, Zuber H. (1996) Isolation, characterization and electron microscopy analysis of a hemidiscoidal phycobilisome type from the cyanobacterium Anabaena sp. PCC 7120. Eur J Biochem. 236:1010-1024.

Duerring M, Huber R, Bode W, Ruembeli R, Zuber H. (1990) Refined three-dimensional structure of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus at 2.7 A. J Mol Biol. 211:633-644.

Duerring M, Schmidt GB, Huber R. (1991) Isolation, crystallization, crystal structure analysis and refinement of constitutive C-phycocyanin from the chromatically adapting cyanobacterium Fremyella diplosiphon at 1.66 A resolution. J Mol Biol. 217:577-592.

Eberlein M, Kufer W. (1990) Genes encoding both subunits of phycoerythrocyanin, a light-harvesting biliprotein from the cyanobacterium Mastigocladus laminosus. Gene. 94:133-136.

Elmorjani K, Thomas JC, Sebban P. (1986) Phycobilisomes of wild type and pigment mutants of the cyanobacterium Synechocystis PCC 6803. Arch Microbiol.146:186-191.

Fairchild CD, Glazer AN. (1994a)Oligomeric structure, enzyme kinetics, and substrate specificity of the phycocyanin alpha subunit phycocyanobilin lyase. J Biol Chem. 269:8686-8694.

Fairchild CD, Glazer AN. (1994b) Nonenzymatic bilin addition to the alpha subunit of an apophycoerythrin. J Biol Chem. 269:28988-28996.

6. References 157

Fairchild CD, Zhao J, Zhou J, Colson SE, Bryant DA, Glazer AN. (1992) Phycocyanin alpha-subunit phycocyanobilin lyase. Proc Natl Acad Sci USA. 89:7017-7021

Falk H. (1989) The Chemistry of Linear Oligopyrroles and Bile Pigments. Wien, New York: Springer.

Falk H. & Müller N. (1982) Force field calculations on linear polypryrrole systems.Tetrahedron 39: 1875-1885.

Ficner R, Lobeck K, Schmidt G, Huber R. (1992) Isolation, crystallization, crystal structure analysis and refinement of B-phycoerythrin from the red alga Porphyridium sordidum at 2.2 A resolution. J Mol Biol. 228:935-950.

Foerstendorf H, Parbel A, Scheer H, Siebert F. Z,E (1997)isomerization of the alpha-84 phycoviolobilin chromophore of phycoerythrocyanin from Mastigocladus laminosus investigated by Fourier-transform infrared difference spectroscopy. FEBS Lett. 402:173-176.

Frank G., Sidler W, Widmer H. & Zuber H. (1978) The complete amino acid sequence of both subunits of C-phycocyanin from the cyanobacterium Mastigocladus laminosus. Hoppe Seylers Z. Physiol. Chem. 359: 1491-1507.

Frankenberg N, Lagarias JC. (2003)Phycocyanobilin:ferredoxin oxidoreductase of Anabaena sp. PCC 7120. Biochemical and spectroscopic characterization. J Biol Chem. 278:9219-9226.

Füglistaller P, Suter F. & Zuber H. (1983) The complete amino-acid sequence of both subunits of phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus. Hoppe Seylers Z. Physiol. Chem. 364: 691-712.

Gambetta GA, Lagarias JC. (2001)Genetic engineering of phytochrome biosynthesis in bacteria. Proc Natl Acad Sci USA . 98:10566-10571.

Gantt E. (1975) Phycobilisomes: Light-harvesting pigment complexes. BioSience 25: 781-788.

Gantt E. (1980) Structure and function of phycobilisomes: light harvesting pigment complexes in red and blue-green algae. Int. Rev. Cyt. 66: 45-80.

Gantt E. (1986) Phycobilisomes. In: Photosynthesis III. Staehelin, L. A. & Arntzen, C. J.(Eds.). pp.327-337. Springer-Verlag, Berlin.

Gantt E. (1988) Phycobilisomes: assessment of core structure and thylakoid interaction. In:Light-Energy Transduction in Photosynthesis: Higher Plants and Bacterial Models.

6. References 158

Stevens,S. E. & Bryant, D. A. (Eds.).pp. 91-101. American Society of Plant Physiologists, Rockeville,USA.

Gindt YM, Zhou J, Bryant DA and Sauer K. (1992) Core mutations of Synechococcus sp. PCC 7002 phycobilisomes:a spectroscopic study. J Photochem Photobiol B 15: 75–89.

Gindt YM, Zhou J, Bryant DA, Sauer K. (1994) Spectroscopic studies of phycobilisome subcore preparations lacking key core chromophores: assignment of excited state energies to the Lcm, beta 18 and alpha AP-B chromophores. Biochim. Biophys. Acta. 1186:153-162.

Glazer AN. (1985) Light harvesting by phycobilisomes. Annu Rev Bioph Biom., 14: 47-77

Glazer AN. (1987) Phycobilisomes: assembly and attachment. In: The Cyanobacteria. Fay,P. & Van Baalen, C. (Eds.).pp. 69-88. Elsevier Biomedical, Amsterdam.

Glazer AN. (1988) Phycobilisomes. Meth. Enzymol. 167: 304-312. Glazer AN. (1989) Light guides. Directional energy transfer in a photosynthetic antenna. J.Biol. Chem. 264: 1-4

Glazer AN. (1994) Adaptive variations in phycobilisome structure. Adv. Mol. Cell Biol. 10:119-149

Glazer A, Fang S & Brown D. (1973) Spectroscopic properties of C-phycocyanin and of its α and β subunits. J. Biol. Chem. 248: 5679-5685.

Glazer, A. N. & Fang, S. (1973) Chromophore content of blue-green algal phycobiliproteins. J. Biol. Chem. 248: 663-671.

Glazer AN and Stryer L. (1983) Fluorescent tandem phycobiliprotein conjugates. Emission wavelength shifting by energy transfer. Biophys J 43: 383–386.

.Glauser M, Bryant DA, Frank G, Wehrli E, Rusconi SS, Sidler W, Zuber H. (1992) Phycobilisome structure in the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC 7120. Eur J Biochem. 205:907-915.

Gottschalk L, Lottspeich F, Scheer H. (1994) Reconstitution of an allophycocyanin trimer complex containing the C-terminal 21-23 kDa domain of the core-membrane linker polypeptide Lcm. Z Naturforsch [C]. 49:331-336.

Grossman AR, Schaefer MR, Chiang GG and Collier JL. (1993) Environmental effects on the light-harvesting complex of cyanobacteria. Microbiol. Rev. 57:725-749

6. References 159

Grubmayr K, Wagner UG. (1988a) Intramolecular nucleophilic addition of 2,3-dihydrodipyrrin-1-(10H)-ones. Monatsh Chem. 119:813-831

Grubmayr K, Wagner UG. (1988b) Mechanism of the nucleophilic addition to 2,3-dihydrodipyrrin-1-(10H)-ones. Monatsh Chem 119:793-812.

Grubmayr K., Wagner UG. (1988c) Addition of thiols to 3-ethylidene-2,3-dihydrodipyrrin-1(10H)-ones - a model study on the covalent chromophore-protein bond in biliproteins. Monatsh Chem. 119:965-983

Guan XY, Qin S, Su ZL, Zhao FQ, Ge BS, Li FC, Tang XX. (2007) Combinational biosynthesis of a fluorescent cyanobacterial holo-α - phycocyanin in Escherichia coli by using one expression vector. Appl. Biochem. Biotech. 142:52-59.

Hagiwara Y, Sugishima M, Takahashi Y & Fukuyama K. (2006) Crystal structure of phycocyanobilin:ferredoxin oxidoreductase in complex with biliverdin IXalpha, a key enzyme in the biosynthesis of phycocyanobilin. Proc. Natl. Acad. Sci. USA 103: 27-32.

He JA., Hu YZ, Jiang LJ.(1996) Photochemistry of phycobiliproteins: first observation of reactive oxygen species generated from phycobiliproteins on photosensitization. J Am Chem Soc. 118:8957-8959

Houmard J, Capuano V, Colombano MV, Coursin T, (1990)Tandeau de Marsac N. Molecular characterization of the terminal energy acceptor of cyanobacterial phycobilisomes. Proc Natl Acad Sci U S A. 87:2152-2156.

Hu IC, Lee TR, Lin HF, Chiueh ChCh, and Lyu PCh (2006) Biosynthesis of Fluorescent Allophycocyanin R-Subunits by Autocatalytic Bilin Attachment, Biochemistry, 45:7092-7099

Huang B, Wang GC, Zeng CK, Li ZG. (2002)The experimental research of R-phycoerythrin subunits on cancer treatment: a new photosensitizer in PDT. Cancer Biother Radiopharm. 17:35-42.

Huang F, Parmryd I, Nilsson F, Persson AL, Pakrasi HB, Andersson B, Norling B. (2002) Proteomics of Synechocystis sp. strain PCC 6803: identification of plasma membrane proteins. Mol Cell Proteom 1:956–996

Hunsucker SW, Klage K, Slaughter SM, Potts M, Helm RF(2004) Biochem Biophys Res Commun 317:1121–1127

Imashimizu M, Fujiwara S, Tanigawa R, Tanaka K, Hirokawa T, Nakajima Y, Higo J, Tsuzuki M. (2003)Thymine at -5 is crucial for cpc promoter activity of Synechocystis sp. strain PCC 6714. J Bacteriol. 185:6477-6480.

6. References 160

Inomata K, Noack S, Hammam MA, Khawn H, Kinoshita H, Murata Y, et al. (2006) Assembly of synthetic locked chromophores with Agrobacterium phytochromes Agp1 and Agp2. J Biol Chem 281: 28162–28173.

Isailovic D, Li H-W, Yeung E.S.(2004) Isolation and characterization of R-phycoerythrin subunits and enzymatic digests. J Chromatogr A. 1051:119-130

Jiang T, Zhang JP, Chang WR, Liang DC. (2001)Crystal structure of R-phycocyanin and possible energy transfer pathways in the phycobilisome. Biophys J. 81:1171-1179.

Jorissen HJ, Quest B, Remberg A, Coursin T, Braslavsky SE, Schaffner K, de Marsac NT, Gartner W. (2002)Two independent, light-sensing two-component systems in a filamentous cyanobacterium. Eur J Biochem. 269:2662-26671.

Jung LJ, Chan CF, Glazer AN. (1995) Candidate genes for the phycoerythrocyanin alpha subunit lyase. Biochemical analysis of pecE and pecF interposon mutants. J Biol Chem. 270:12877-12884.

Kahn K, Mazel D, Houmard J, Tandeau de Marsac N, Schaefer MR. (1997)A role for cpeYZ in cyanobacterial phycoerythrin biosynthesis. J Bacteriol. 179(4):998-1006.

Kahn K, Schaefer MR. (1997)rpbA controls transcription of the constitutive phycocyanin gene set in Fremyella diplosiphon. J Bacteriol. 179:7695-704.

Kaiser A, Hammels I, Gottwald A N, Assar M, Zaghloul MS, Motaal BA, Hauber J, Hoerauf A. (2007) Modification of eukaryotic initiation factor 5A from Plasmodium vivax by a truncated deoxyhypusine synthase from Plasmodium falciparum: An enzyme with dual enzymatic properties. Bioorganic & Med. Chem. 15:6200-6207.

Kaiser A, Ulmer D, Goebel T, Holzgrabe U, Saeftel M, Hoerauf A. (2006) Inhibition of hypusine biosynthesis in Plasmodium: a possible, new strategy in prevention and therapy of malaria. Mini-Rev. Med. Chem. 6:1231-1241

Klotz AV. & Glazer AN. (1985) Characterization of the bilin attachment sites in R-phycoerythrin. J Biol Chem 260: 4856-4863.

Klotz AV, and Glazer AN. (1987) Gamma-N-methylasparagine in phycobiliproteins. Occurrence, location and biosynthesis. J. Biol. Chem. 262:17350–17355.

Klotz AV, Leary JA and Glazer AN (1986) Post-translational methylation of asparaginyl residues. Identification of β-71 γ-N-methylasparagine in allophycocyanin. J. Biol. Chem. 261:15891–15894.

Klotz AV, Thomas BA, Glazer AN and. Blacher RW. (1990). Detection of methylated

6. References 161

asparagines and glutamine residues in polypeptides. Anal.Biochem. 18:95–100.

Kohchi T, Kataoka H, Linley PJ. (2005) Biosynthesis of chromophores for phytochrome and related photoreceptors. Plant Biotech (Tokyo, Japan) 22:409-413.

Kohchi T, Mukougawa K, Frankenberg N, Masuda M, Yokota A and Lagarias JC. (2001) The Arabidopsis HY2 gene encodes phytochromobilin synthase, a ferredoxindependent biliverdin reductase. Plant Cell 13: 425–436.

Köst HP, Rüdiger W and Chapman DJ. (1975) Über die Bindung zwischen Chromophor und Protein in Biliproteiden. 1. Abbauversuche und Spektraluntersuchungen. Liebig’s Ann Chem 1975: 1582–1593.

Kufer W & Björn GS. (1989) Photochromism of the cyanobacterial light harvesting biliprotein phycoerythrocyanin. Physiol. Plant 75: 389-394.

Kupka M, Scheer H. (2008) Unfolding of C- phycocyanin followed by loss of non-covalent chromophore-protein interactions 1. Equilibrium experiments. Biochim. Biophys. Acta. 1777:94-103.

Lagarias JC, Glazer AN, Rapoport H. (1979) Chromopeptides from C-phycocyanin. structure and linkage of a phycocyanobilin bound to the β subunit. J. Am. Chem. Soc. 101: 5030-5037.

Lagarias JC, Klotz AV, Dallas JL, Glazer AN, Bishop JE, Connel JF, Rapoport H.(1988) Exclusive A-ring Linkage for singly attached phycocyaninbilins and phycoerythrobilins in phycobiliproteins.J Biol Chem 263:12977-12985

Lamparter T. (2004) Evolution of cyanobacterial and plant phytochromes. FEBS Lett 573: 1–5.

Lamparter T and Michael N. (2005) Agrobacterium phytochrome as an enzyme for the production of ZZE bilins.Biochemistry 44: 8461–8469.

Lamparter T, Michael N, Mittmann F, Esteban B. (2002) Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site. Proc Natl Acad Sci USA. 99:11628-11633.

Lamparter T, Carrascal M, Michael N, Martinez E, Rottwinkel G., Abian J.(2004) The Biliverdin Chromophore Binds Covalently to a Conserved Cysteine Residue in the N-Terminus of Agrobacterium Phytochrome Agp1. Biochemistry. 43:3659-3669.

Li H, Sherman LA. (2002)Characterization of Synechocystis sp. strain PCC 6803 and deltanbl mutants under nitrogen-deficient conditions. Arch Microbiol. 178:256-266.

6. References 162

Li L, Lagarias JC. (1994)Phytochrome assembly in living cells of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 91:12535-12539.

Liu JY, Jiang T, Zhang JP, Liang DC. (1999) Crystal structure of allophycocyanin from red algae Porphyra yezoensis at 2.2-A resolution. J Biol Chem. 274:16945-16952.

Lundell DJ, Glazer AN. (1981) Allophycocyanin B. A common β subunit in Synechococcus allophycocyanin B (λmax 670 nm) and allophycocyanin (λmax 650 nm), J. Biol. Chem. 256: 12600-12606.

Luque I, Ochoa De Alda JA, Richaud C, Zabulon G, Thomas JC, Houmard J. (2003)The NblAI protein from the filamentous cyanobacterium Tolypothrix PCC 7601: regulation of its expression and interactions with phycobilisome components. Mol Microbiol. 50:1043-54.

Ma Y, Xie J, Zhang C and Zhao J. (2007) Three-stage refolding/unfolding of the dual-color b-subunit in R-phycocyanin from Polysiphonia urceolata. Biochem Biophys Res Commun 352: 787-793.

MacColl R. (1998) Cyanobacterial phycobilisomes J Struct Biol. 124:311-334.

MacColl R. (2004) Allophycocyanin and energy transfer. Biochim. Biophys. Acta. 1657:73-81.

MacColl R, Lam I, Choi CY, Kim J. (1994) Exciton splitting in phycoerythrin 545. J Biol Chem. 269:25465-25469.

MacColl R, Malak H, Gryczynski I, Eisele LE, Mizejewski GJ, Franklin E, Sheikh H, Montellese D, Hopkins S, MacColl LC. (1998) Phycoerythrin 545: monomers, energy migration, bilin topography, and monomer/dimer equilibrium. Biochemistry. 37:417-423.

Migita CT, Zhang X, Yoshida T. (2003)Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. Properties of the heme complex of recombinant active enzyme. Eur J Biochem. 270:687-698.

Miller CA, Leonard HS, Pinsky IG, Turner BM, Williams SR, Jr LH, Fletcher AF, Shen G, Bryant DA, Schluchter WM.(2008) Biogenesis of Phycobiliproteins III. CpcM is the asparagine methyltransferase for phycobiliprotein β-subunit in cyanobacteria. J Biol Chem 283:19293-19300

Miller CG, Strauch KL, Kukral AM, Miller JL, Wingfield PT,Mazzei GJ, Werlen RC, Graber P, and Movva NR. (1987) N-terminal methionine-specific peptidase in Salmonella typhimurium.Proc. Natl. Acad. Sci. USA. 84:2718–2722

6. References 163

Mimuro M, Fueglistaller P, Ruembeli R, Zuber H. (1986) Functional assignment of chromophores and energy transfer in C phycocyanin isolated from the thermophilic cyanobacterium Mastigocladus laminosus. Biochim. Biophys. Acta. 848: 155-166

Minami Y, Yamada F, Hase T, Matsubara H, Murakami A, Fujita Y, Takao T. and Shimonishi Y. (1985) Amino acid sequences of allophycocyanin α- and β-subunits isolated from Anabaena cylindrica: presence of an unknown derivative of aspartic acid in the β-subunit. FEBS Lett. 191:216–220.

Montgomery BL, Casey ES, Grossman AR, Kehoe DM (2004) AplA, a Member of a New Class of Phycobiliproteins Lacking a Traditional Role in Photosynthetic Light Harvesting. J Bacteriol 186:7420–7428.

Muramoto T, Tsurui N, Terry MJ, Yokota A, Kohchi T. (2002)Expression and biochemical properties of a ferredoxin-dependent heme oxygenase required for phytochrome chromophore synthesis. Plant Physiol. 130:1958-1966.

Nagy JO, Bishop JE, Klotz AV, Glazer AN & Rapoport H. (1985) Bilin attachment sites in the alpha, beta, and gamma subunits of R-phycoerythrin. Structural studies on singly and doubly linked phycourobilins. J. Biol. Chem. 260: 4864-4868.

Nakajima M, Sakamoto T, Wada K. (2002) The complete purification and characterization of three forms of ferredoxin-NADP(+) oxidoreductase from a thermophilic cyanobacterium Synechococcus elongatus. Plant Cell Physiol. 43:484-493.

Nakamura Y, Kaneko T, Hirosawa M, Miyajima N & Tabata S. (1998) Extension of CyanoBase. CyanoMutants: repository of mutant information on Synechocystis sp. strain PCC6803 Nucleic Acids Res. 26:63–67.

Nanni B, Balestreri E, Dainese E, Cozzani I, Felicioli R. (2001) Characterisation of a specific phycocyanin-hydrolysing protease purified from Spirulina platensis.Microbiol Res. 156:259-266.

Nomsawai P, Tandeau de Marsac N, Thomas JC, Tanticharoen M, Cheevadhanarak S (1999) Light regulation of phycobilisome structure and gene expression in Spirulina platensis C1 (Arthrospira sp. PCC 9438).Plant Cell Physiol 40:1194–1202

Noubir S, Luque I, Ochoa de Alda JA, Perewoska I, Tandeau de Marsac N, Cobley JG, Houmard J. (2002)Co-ordinated expression of phycobiliprotein operons in the chromatically adapting cyanobacterium Calothrix PCC 7601: a role for RcaD and RcaG. Mol Microbiol. 43:749-762.

Ong LJ, Glazer AN. (1987) R-phycocyanin II, a new phycocyanin occurring in marine Synechococcus species. Identification of the terminal energy acceptor bilin in phycocyanins.

6. References 164

J. Biol. Chem. 262: 6323-6327.

Ong LJ, Glazer AN. (1991)Phycoerythrins of marine unicellular cyanobacteria. I. Bilin types and locations and energy transfer pathways in Synechococcus spp. phycoerythrins. J Biol Chem. 266:9515-9527.

Padyana AK, Bhat VB, Madyastha KM, Rajashankar KR, Ramakumar S. (2001)Crystal structure of a light-harvesting protein C-phycocyanin from Spirulina platensis. Biochem Biophys Res Commun. 282:893-898.

Papenbrock J, Grimm B. (2001) Regulatory network of tetrapyrrole biosynthesis--studies of intracellular signalling involved in metabolic and developmental control of plastids. Planta. 213:667-681.

Parbel A, Zhao KH, Breton J, Scheer H. (1997) Chromophore assignment in phycoerythrocyanin from Mastigocladus laminosus. Photosynth. Res. 54: 25-34.

Piven Irina (2006) Characterisation of mechanisms and components of protein phosphorylation in photosynthetic membranes of Synechocystis sp. PCC 6803. Ph.D. thesis, University of Munich, Germany

Pizarro SA, Sauer K. (2001) Spectroscopic study of the light-harvesting protein C-phycocyanin associated with colorless linker peptides. Photochem Photobiol. 73:556-563.

Pueyo JJ, Go′ mez-Moreno C (1991) Purification of ferredoxin-NADP+ reductase, ferredoxin and flavodoxin from a single batch of the cyanobacterium Anabaena PCC7119.Prep Biochem 21:191–204

Quest B, Gartner W. (2004)Chromophore selectivity in bacterial phytochromes: dissecting the process of chromophore attachment. Eur J Biochem. 271:1117-1126.

Reuter W, Nickel-Reuter C (1993) Molecular assembly of the phycobilispomes from the cyanobacterium Mastigocladus laminosus.J Photochem Photobiol B Biol 18:51–66

Reuter W, Westermann M, Brass S, Ernst A, Boger P, Wehrmeyer W. (1994) Structure, composition, and assembly of paracrystalline phycobiliproteins in Synechocystis sp. strain BO 8402 and of phycobilisomes in the derivative strain BO 9201. J Bacteriol. 176:896-904.

Reuter W, Wiegand G, Huber R, Than ME. (1999)Structural analysis at 2.2 A of orthorhombic crystals presents the asymmetry of the allophycocyanin-linker complex, AP.LC7.8, from phycobilisomes of Mastigocladus laminosus. Proc Natl Acad Sci USA. 96:1363-1368.

6. References 165

Rhie G, Beale SI. (1995)Phycobilin biosynthesis: reductant requirements and product identification for heme oxygenase from Cyanidium caldarium. Arch Biochem Biophys. 320:182-194.

Ritter S, Hiller RG, Wrench PM, Welte W, Diederichs K. (1999) Crystal structure of a phycourobilin-containing phycoerythrin at 1.90-A resolution. J Struct Biol. 126:86-97.

Rommàn RB, Alv à rez-Pez, JM, Aci é n Fernàndez, F.G. and Molina Grima, E. (2002) Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum. J. Biotechnol. 93: 73-85

Ruembeli R, Wirth M, Suter F, Zuber H (1987) The phycobiliprotein beta 16.2 of the allophycocyanin core from the cyanobacterium Mastigocladus laminosus. Characterization and complete amino-acid sequence Biol Chem Hoppe–Seyler 368:1–9.

Sauer, Kenneth and Scheer, Hugo (1988) Excitation transfer in C-phycocyanin. Förster transfer rate and exciton calculations based on new crystal structure data for C-phycocyanins from Agmenellum quadruplicatum and Mastigocladus laminosus. Biochim. Biophys. Acta. 936: 157-170.

Saunée NA, Williams SR, Bryant DA, and Schluchter WM. (2008) Biogenesis of Phycobiliproteins II. CpcS-I and CpcU comprise the heterodimeric bilin lyase that attachs phycocyanobilin to Cys-82 of β-phycocyanin and Cys-81 of allophycocyanin subunits in synechococcus sp. PCC 7002.J. Biol. Chem. 283:7513-7522

Sawatzki J, Fischer R, Scheer H., Siebert F.(1990) Fourier transform Raman spectroscopy applied to biological systems. Proc. Natl. Acad. Sci. USA 87:5903-5906

Sawaki H, Sugiyama T, Omata T. (1998) Promoters of the phycocyanin gene clusters of the cyanobacterium Synechococcus sp. strain PCC 7942. Plant Cell Physiol. 39:756-61.

Scharnagl C, Schneider S. (1991)UV visible absorption and circular dichroism spectra of the subunits of C- phycocyanin. I: Quantitative assessment of the effect of chromophore-protein interaction in the β -subunit. J Photochem. Photobiol.B: Bio., 3:603-614.

Scharnagl C, Schneider S. (1991) UV-visible absorption and circular dichroism spectra of the subunits of C- phycocyanin. II: a quantitative discussion of the chromophore-protein and chromophore-chromophore interactions in the β-subunit. J Photochem. Photobiol. B:Bio., 8:129-157.

Schechter I, Berger A. (1967) On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 27:157–162

6. References 166

Scheer H. (1982) Phycobiliproteins: Molecular aspects of photosynthetic antenna systems.In Light Reaction Path of Photosynthesis. Fong, F.K. (ed). Berlin: Springer , pp. 7-45.

Scheer H, and Zhao KH. (2008) Biliprotein maturation: the chromophore attachment. Mol. Microbiol. 68:263-276.

Schirmer T, Bode W and Huber R. (1987) Refined threedimensional structures of two cyanobacterial C-phycocyanins at 2.1. and 2.5 A resolution – a common principle of phycobilin–protein interaction. J Mol Biol 196:677–695

Schluchter WM, Bryant DA. (1992)Molecular characterization of ferredoxin-NADP+ oxidoreductase in cyanobacteria: cloning and sequence of the petH gene of Synechococcus sp. PCC 7002 and studies on the gene product. Biochemistry.31:3092-3102.

Schluchter WM and Bryant DA. (2002) Analysis and reconstitution of phycobiliproteins. methods for the characterization of bilin attachment reactions. In Heme, Chlorophyll,and Bilins. Smith, A.G., and Witty, M. (eds). Totowa,NJ: Humana Press, pp. 311–334.

Schluchter WM and Glazer AN. (1999) Biosynthesis of phycobiliproteins in cyanobacteria. In The Phototrophic Prokaryotes. Peschek, G.A., Löffelhardt, W., and Schmetterer,G. (eds). New York: Kluwer/Plenum Press, pp.83–95.

Schmidt M, Krasselt A & Reuter W. (2006) Local protein flexibility as a prerequisite for reversible chromophore isomerization in alpha-phycoerythrocyanin. Biochim. Biophys. Acta.1764: 55-62.

Shen G., Leonard HS, Schluchter WM, Bryant DA. (2008b) CpcM Posttranslationally Methylates Asparagine-71/72 of Phycobiliprotein Beta Subunits in Synechococcus sp. Strain PCC 7002 and Synechocystis sp. Strain PCC 6803. J. Bacteriol.190: 4808-4817

Shen G, Saunee NA, Gallo E, Begovic Z, Schluchter WM & Bryant DA. (2004)Identification of novel phycobiliprotein lyases in cyanobacteria. In: Photosynthesis 2004 lightharvesting systems workshop. Niederman, R. A., Blankenship, R. E., Frank, H., Robert, B. & van Grondelle, R. (Eds.). pp.14-15. Saint Adele, Québec, Canada.

Shen G, Saunee N A, Williams SR, Gallo EF, Schluchter WM & Bryant DA.(2006) Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta subunit of phycocyanin in Synechococcus sp. PCC 7002. J. Biol. Chem. 281: 17768-17778.

Shen G., Schluchter WM and Bryant DA. (2008) Biogenesis of Phycobiliproteins I. cpcS-I and cpcU mutants of the cyanobacterium synechococcus sp. PCC 7002 define a heterodimeric phycocyanobilin lyase specific for β-phycocyanin and allophycocyanin subunits. J. Biol. Chem. 283:7503–7512

6. References 167

Sidler WA. (1994). Phycobilisome and phycobiliprotein structures. In Bryant D. A. (Ed), The Molecular Biology of Cyanobacteria. Dordrecht, Netherlands: Kluwer Academic Publishers.

Sidler W, Gysi J, Isker E & Zuber H. (1981) The complete amino acid sequence of both subunits of allophycocyanin, a light harvesting protein-pigment complex from the cyanobacterium Mastigocladus laminosus. Hoppe Seylers Z. Physiol. Chem. 362: 611-628.

Sidler W, Kumpf B, Rüdiger W & Zuber H. (1986) The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon. Biol. Chem. Hoppe Seyler 367: 627-642.

Siebzehnrübl S. (1990) Chromophorzuordnung und reversible Photochemie von C-Phycocyanin und Phycoerythrocyaninen. Dissertation, LMU München.

Six C, Thomas JC, Thion L, Lemoine Y, Zal F & Partensky F. (2005) Two novel phycoerythrin-associated linker proteins in the marine cyanobacterium Synechococcus sp.strain WH8102. J. Bacteriol. 187: 1685-1694.

Spolare P, Joannis CC, Duran E, Isambert A.(2006) commercial applications of microalgae.J Biosci. Bioeng.101:87-96

Storf M, Parbel A, Meyer M, Strohmann B, Scheer H, Deng MG, Zheng M, Zhou M, Zhao KH. (2001) Chromophore Attachment to Biliproteins : Specificity of PecE/PecF, a Lyase -Isomerase for the Photoactive 31-Cys- 84-phycoviolobilin Chromophore of Phycoerythrocyanin. Biochemistry. 40: 12444-12456.

Storf M. (2003) Chromophorbindung und Photochemie der -Untereinheit des Phycoerythrocyanins aus Mastigocladus laminosus. Dissertation, LMU München.

Studier FW & Moffat BA. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113-130.

Stumpe H, Mueller N, Grubmayr K. (1993) Addition of methyl-2-mercaptoacetate to phycocyanobilin dimethyl ester: a model reaction for biliprotein biosynthesis? Tetrahedron Lett, 34:4165-4168.

Sun L, Wang S (2003) Allophycocyanin complexes from the phycobilisome of a thermophilic blue-green alga Myxosarcina concinna Printz. J Photochem Photobiol B Biol 72:45–53.

Swanson RV, Glazer AN. (1990) Separation of phycobiliprotein subunits by reverse-phase high pressure liquid chromatography. Anal Biochem 188:295–299.

Swanson RV, Glazer AN.(1990) Phycobiliprotein methylation: effect of the γ-N-methylasparagine residue on energy transfer in phycocyanin and the phycobilisome. J.

6. References 168

Mol. Biol. 214:787–796.

Swanson RV, Ong LJ, Wilbanks SM, Glazer AN. (1991)Phycoerythrins of marine unicellular cyanobacteria. II. Characterization of phycobiliproteins with unusually high phycourobilin content. J Biol Chem. 266:9528-9534.

Swanson RV, Zhou J, Leary JA, Williams T, de Lorimier R, Bryant DA, Glazer AN. (1992)Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment. J Biol Chem. 267:16146-16154.

Toole CM, Plank TL, Grossman AR, Anderson LK. (1998)Bilin deletions and subunit stability in cyanobacterial light-harvesting proteins. Mol Microbiol. 30:475-486.

Tooley AJ, Cai YA, Glazer AN. (2001)Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host. Proc Natl Acad Sci USA. 98:10560-10565.

Tooley AJ, Glazer AN. (2002)Biosynthesis of the cyanobacterial light-harvesting polypeptide phycoerythrocyanin holo-alpha subunit in a heterologous host. J Bacteriol. 184:4666-4671.

Tu JM, Kupka M, Böhm S, Plöscher M, Eichacker L, Zhao KH and Scheer H.(2008) Intermediate binding of phycocyanobilin to the lyase, CpeS1, and transfer to apoprotein. Photosynth. Res. 95: 163-168.

Tu Sh-L, Sughrue W, David BR, and Lagarias JC. (2006) A Conserved Histidine-Aspartate Pair Is Required for Exovinyl Reduction of Biliverdin by a Cyanobacterial Phycocyanobilin:Ferredoxin Oxidoreductase, J. Biol. Chem., 281: 3127-3136

Viskari PJ, Colyer CL (2002) Separation and quantitation of phycobiliproteins using phytic acid in capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A 972:269–276

Viskari PJ, Kinkade CS, Colyer CL (2001) The determination of phycobiliproteins by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis 22:2327–2335

Wagner JR, Brunzelle JS, Forest KT & Vierstra RD. (2005) A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome. Nature 438:325-331.

Walter NG, Huang CY, Manzo AJ, Sobhy MA. (2008)Do-it-yourself guide: how to use the modern single-molecule toolkit.Nat. Method 5:475-489

6. References 169

Wang XQ, Li LN, Chang WR, Zhang JP, Gui LL, Guo BJ and Liang DC. (2001) Structure of C-phycocyanin from Spirulina platensis at 2.2 Angstrom resolution: a novel monoclinic crystal form for phycobiliproteins in phycobilisomes. Acta Cryst D Biol Cryst 57: 784–792.

Wiegand G, Parbel A, Seifert MH, Holak TA, Reuter W. (2002) Purification, crystallization, NMR spectroscopy and biochemical analyses of alpha-phycoerythrocyanin peptides. Eur J Biochem. 269:5046-5055.

Wu Shu-Hsing, Lagarias JC. (2000) Defining the Bilin Lyase Domain: Lessons from the Extended Phytochrome Superfamily.Biochemistry. 39:13487-13495.

Yang Yu, Ge BS, Guan XY, Zhang WJ, Qin S. (2008) Combinational biosynthesis of a fluorescent cyanobacterial holo-α- allophycocyanin in Escherichia coli. Biotech. Lett. 30:1001-1004.

Zehetmayer P, Hellerer T, Parbel A, Scheer H, Zumbusch A. (2002)Spectroscopy of single phycoerythrocyanin monomers: dark state identification and observation of energy transfer heterogeneities. Biophys J. 83:407-415.

Zehetmayer P, Kupka M, Scheer H, Zumbusch A. (2004)Energy transfer in monomeric phycoerythrocyanin. Biochim. Biophys. Acta. 1608:35-44.

Zhang SP, Qian SP, Zhao JQ, Yao SD, Jiang LJ. (1999)Characterization of the transient species generated by the photoexcitation of C-phycocyanin from Spirulina platensis: a laser photolysis and pulse radiolysis study. Biochim. Biophys. Acta. 1472:270-278.

Zhang S, Xie J, Zhang J, Zhao J, Jiang L. (1999)Electron spin resonance studies on photosensitized formation of hydroxyl radical by C-phycocyanin from Spirulina platensis. Biochim. Biophys. Acta. 1426:205-211.

Zhang SP, Zhao JQ, Jiang LJ. (2000)Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions. Free Radic Res. 33:489-496.

Zhao FQ and Qin S(2006) Evolutionary Analysis of Phycobiliproteins: Implications for Their Structural and Functional Relationships. J. Mol. Evol., 63:330–340

Zhao J, Shen G, Bryant DA. (2001)Photosystem stoichiometry and state transitions in a mutant of the cyanobacterium Synechococcus sp. PCC 7002 lacking phycocyanin. Biochim. Biophys. Acta.1505:248-257.

Zhao KH, Haessner R, Cmiel E, and Scheer H. (1995) Type I reversible of phycoerythrocyanin involves Z/E-isomerization of α-84 phycoviolobilin chromophore. Biochim. Biophys. Acta. 1228: 235-243.

6. References 170

Zhao KH & Scheer H. (1999) Intermediates of reversible photochemistry of phycoerythrocyanin α-subunit from Mastigocladus laminosus probed by low temperature absorption and cirular dichroism spectroscopy. Int. J. Photoenergy 1: 25-30.

Zhao KH, Deng MG, Zheng M, Zhou M, Parbel A, Storf M, Meyer M, Strohmann B & Scheer H. (2000) Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by PecE and PecF.FEBS Lett. 469: 9-13.

Zhao KH, Ran Y, Li M, Sun YN, Zhou M, Storf M, Kupka M, Böhm S, Bubenzer C and Scheer H. (2004a) Photochromic biliproteins from the cyanobacterium Anabaena sp. PCC 7120: lyase activities, chromophore exchange and photochromism in phytochrome and phycoerythrocyanin. Biochemistry 43: 11576-11588.

Zhao KH, Su P, Böhm S, Song B, Zhou M, Bubenzer C & Scheer H. (2005a) Reconstitution of phycobilisome core-membrane linker, Lcm, by autocatalytic chromophore binding to ApcE. Biochim. Biophys. Acta. 1706: 81-87.

Zhao KH, Su P, Li J, Tu JM, Zhou M, Bubenzer C & Scheer H. (2006a) Chromophore attachment to phycobiliprotein beta-subunits: phycocyanobilin: cysteine-beta84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena sp.PCC7120. J. Biol. Chem. 281: 8573-8581.

Zhao KH, Su P, Tu JM, Wang X, Liu H, Plöscher M, Eichacker L, Yang B, Zhou M and Scheer H (2007a) Phycobilin:cysteine-84 biliprotein lyase,a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins. Proc Natl Acad Sci USA, 104:14300-14305.

Zhao KH, Wu D, Zhou M, Zhang L, Böhm S, Bubenzer C & Scheer H. (2005b)Amino acid residues associated with enzymatic activity of the isomerizing phycoviolobilinlyase PecE/F. Biochemistry 44: 8126-8137.

Zhao KH, Wu D, Wang L, Zhou M, Storf M, Bubenzer C, Strohmann B & Scheer H. (2002) Characterization of Phycoviolobilin Phycoerythrocyanin-α84-cystein-lyase-(isomerizing) from Mastigocladus laminosus. Eur. J. Biochem. 269: 4542-4550.

Zhao KH, Wu D, Zhang L, Zhou M, Böhm S, Bubenzer C & Scheer H. (2006b)Chromophore attachment in phycocyanin. Functional amino acids of phycocyanobilin:α- hycocyanin lyase and evidence for chromophore binding. FEBS J. 273: 1262-1274.

Zhao KH, Zhang J, Tu JM, Böhm S, Plöscher M, Eichacker L, Bubenzer C,Scheer H, Wang X, and Zhou M. (2007b) Lyase activities of CpcS and CpcT-like proteins from Nostoc sp. PCC7120, and sequential reconstitution of binding sites of phycoerythrocyanin and phycocyanin ß-subunits. J. Biol. Chem. 282: 34093-34103.

6. References 171

Zhao KH, Zhu JP, Deng MG, Zhou M, Storf M, Parbel A & Scheer H. (2003) Photochromic chromopeptides derived from phycoerythrocyanin: biophysical and biochemical characterization. Photochem. Photobiol. Sci. 2: 741-748.

Zhao KH, Zhu JP, Song B, Zhou M, Storf M, Böhm S, Bubenzer C & Scheer H.(2004b) Non-enzymatic chromophore attachment in biliproteins: Conformational control by the detergent Triton X-100. Biochim. Biophys. Acta 1657: 131-145.

Zhou J, Gasparich GE, Stirewalt VL, de Lorimier R & Bryant DA. (1992) The cpcE and cpcF genes of Synechococcus sp. PCC 7002. J. Biol. Chem. 23: 16138-16145.

Zolla L, Bianchetti M (2001) High-performance liquid chromatography coupled on-line with electrospray ionization mass spectrometry for the simultaneous separation and identification of the Synechocystis PCC 6803 phycobilisome proteins. J Chromatogr A 912:269–279

Zuber H. (1987) The structure of light-harvesting pigment-protein complexes. In: The Light Reactions. Barber, J. (Eds.).pp..157-259. Elsevier Biomedical, Amsterdam.

Curriculum Vitae

Personal data

Family name: Tu

First name: Jun-Ming

Data of birth: 10.02.1972

Place of birth: Hubei, P.R. China

Family status: married

Education

2006- 2008 Post-graduate study in the

Ludwig-Maximilians-University, Department

of Biology I, Botanic, Munich,Germany

2005- 2006 Post-graduate study in the Huazhong

University of Science and Technology,

College of Environmental Science and

Engineering, Wuhan, P.R. China.

1998-2000 Master study in Jiangnan University, School

of Biotechnology,Wuxi,P.R. China

1989- 1993 Bachelor of Fermentation Engineering,

Hubei University of Technology, Wuhan, P.R.

China

1983-1989 Secondary School, Ezhou, P.R. China

Eidesstattliche Erklärung Hiermit bestätige ich, dass ich die vorliegende Arbeit selbständig verfasst und

keine anderen als die hier angegebenen Quellen und Hilfsmittel verwendet

habe. Ich versichere ferner, dass ich die vorliegende Arbeit nicht schon als

Doktor-, Magister oder Diplomarbeit bei einer anderen Hochschule eingereicht

habe. Ort, Datum Unterschrift

Documents

Phycobiliprotein Lyases: Structure of Reconstitution